Immortalization and malignant transformation of eucaryotic cells

This review is aimed at understanding the mechanisms of cell immortalization by different «immortalizing agents», oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor «oncogene and chromosome addiction», i...

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Datum:	2012
Hauptverfasser:	Stepanenko, O.A., Kavsan, V.M.
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Sprache:	English
Veröffentlicht:	Інститут клітинної біології та генетичної інженерії НАН України 2012
Schriftenreihe:	Цитология и генетика
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spelling	irk-123456789-1264632017-11-25T03:02:58Z Immortalization and malignant transformation of eucaryotic cells Stepanenko, O.A. Kavsan, V.M. Обзорные статьи This review is aimed at understanding the mechanisms of cell immortalization by different «immortalizing agents», oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor «oncogene and chromosome addiction», intra-/intertumor heterogeneity, and chromosome instability. Целью настоящего обзора является понять механизмы клеточной иммортализации различными «иммортализующими агентами» ,онкоген-индуцируемой клеточной трансформации иммортализированных клеток и умеренный ответ на терапию из-за «склонности» опухоли к приобретению многочисленных генных и хромосомных изменений, внутри- и межопухолевой гетерогенности. Мета даного огляду зрозуміти механізми клітинної іморталізації різними «іморталізуючими агентами» ,онкогеніндукованої клітинної трансформації іморталізованих клітин і помірну відповідь на терапію через «схильність» пухлини до придбання численних генних та хромосомних змін та гетерогенністю усередині і між пухлинами. 2012 Article Immortalization and malignant transformation of eucaryotic cells / O.A. Stepanenko, V.M. Kavsan // Цитология и генетика. — 2012. — Т. 46, № 2. — С. 36-75. — Бібліогр.: 413 назв. — англ. 0564-3783 http://dspace.nbuv.gov.ua/handle/123456789/126463 576.385.5 en Цитология и генетика Інститут клітинної біології та генетичної інженерії НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Обзорные статьи Обзорные статьи
spellingShingle	Обзорные статьи Обзорные статьи Stepanenko, O.A. Kavsan, V.M. Immortalization and malignant transformation of eucaryotic cells Цитология и генетика
description	This review is aimed at understanding the mechanisms of cell immortalization by different «immortalizing agents», oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor «oncogene and chromosome addiction», intra-/intertumor heterogeneity, and chromosome instability.
format	Article
author	Stepanenko, O.A. Kavsan, V.M.
author_facet	Stepanenko, O.A. Kavsan, V.M.
author_sort	Stepanenko, O.A.
title	Immortalization and malignant transformation of eucaryotic cells
title_short	Immortalization and malignant transformation of eucaryotic cells
title_full	Immortalization and malignant transformation of eucaryotic cells
title_fullStr	Immortalization and malignant transformation of eucaryotic cells
title_full_unstemmed	Immortalization and malignant transformation of eucaryotic cells
title_sort	immortalization and malignant transformation of eucaryotic cells
publisher	Інститут клітинної біології та генетичної інженерії НАН України
publishDate	2012
topic_facet	Обзорные статьи
url	http://dspace.nbuv.gov.ua/handle/123456789/126463
citation_txt	Immortalization and malignant transformation of eucaryotic cells / O.A. Stepanenko, V.M. Kavsan // Цитология и генетика. — 2012. — Т. 46, № 2. — С. 36-75. — Бібліогр.: 413 назв. — англ.
series	Цитология и генетика
work_keys_str_mv	AT stepanenkooa immortalizationandmalignanttransformationofeucaryoticcells AT kavsanvm immortalizationandmalignanttransformationofeucaryoticcells
first_indexed	2025-07-09T05:04:25Z
last_indexed	2025-07-09T05:04:25Z
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fulltext	36 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 © O.A. STEPANENKO, V.M. KAVSAN, 2012 UDK 576.385.5 A.A. STEPANENKO, V.M. KAVSAN Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv E-mail: kavsan@imbg.org.ua IMMORTALIZATION AND MALIGNANT TRANSFORMATION OF EUKARYOTIC CELLS The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortal- ized cells never have the normal diploid karyotype, nev- ertheless, they cannot grow over one another in cell cul- ture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromo- some changes. Multiple genetic rearrangements, includ- ing whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (onco- genes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC- F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different «immortalizing agents», oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor «oncogene and chromosome addiction», intra-/intertumor heterogeneity, and chromosome instability. Introduction. Malignant transformation is the process by which cells acquire the properties of cancer. The first successful malignant transfor- mation in vitro was achieved with the polyoma virus on Syrian hamster embryo cells, followed by transformation with chemical carcinogens in the mid-1960th (reviewed in [1]). Reports of hu- man cell transformation using viruses and viral oncogenes appeared only in the late 1970th [1]. In early 80th it was shown that immortalized NIH3T3 mouse fibroblast cells introduced with total ge- nomic DNA from human tumors were converted into cancer cells; later it was found that H-RAS gene harboring a point mutation induced trans- formation of NIH3T3 cells in culture and con- ferred on them the ability to induce tumors in nude mice (reviewed in [2]). These discoveries marked an advent of the intense searching for the abnormal genes influencing the development of human cancer that continues today [3]. Early works stated that in vitro transformation of human cells by a single carcinogenic agent in contrast to rodent cells was an extraordinarily rare event [1]. Moreover, a spontaneous im- mortalization following senescence was also an extremely rare event in human fibroblasts and epithelial cells, although it occurred commonly in rodent cells with varying frequencies depend- ing on species from which the cells were derived [1, 4]. One of the explanations of intrinsic anti- neoplastic mechanisms of human cells was dif- ferences in telomere biology between human and murine cells. Mouse cells begin their replication ex vivo with extremely long telomeres: 3–10-fold longer than in identical human cells and the ten- dency for progressive telomere erosion might ef- fectively be countered by the basal telomerase ac- tivity that is constitutively present in mouse cells [1, 4–8]. Additionally, the basal metabolic rate is about 7-fold higher in mice than in humans and this affects the levels of endogenous oxidants and other mutagens that are produced as by-products of normal oxidative metabolism resulting in 18- fold more breakdown products of DNA in mice [4]. Moreover, the rates of metabolic conversion of procarcinogens to carcinogens and the detoxi- fication of many other potential mutagens can occur with greatly differing kinetics [4]. Further- more, humans have more efficient DNA repair system, and the rate of 5-methylcytosine decline during cellular senescence is much slower in hu- man cells than in mouse cells [1]. Îáçîðíûå ñòàòüè 37ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells Nevertheless, telomerase-deficient primary mouse embryonic fibroblasts (MEFs) could be immortalized/transformed in culture, and gener- ated tumors in nude mice following transforma- tion [9]. It was concluded that telomerase is not required for establishment of immortalized cell lines, oncogenic transformation, or tumor forma- tion in mice. Another research group transformed human primary fibroblasts and human primary mesodermal cells introducing simultaneously three oncogenes E1A, MDM2, and H-RASV12. These cells formed colonies in soft agar and tumors in mice, but they and the majority of the tumors derived from them lacked telomerase activity, and telomere erosion was observed [10]. Authors have deduced that telomere maintenance is not obligatory for tumorigenic conversion. To the point, human primary melanomas show telomere maintenance as a late event in tumor progression (metastatic melanoma), thus, telomere mainte- nance/immortalization is associated with pro- gression rather than initiation of melanoma [11]. Furthermore, like primary human cells, pri- mary MEFs require combination of two «hits» to acquire the capacity to form tumors [9, 12- 19]. There are also cases of a conversion of nor- mal primary rodent [20–23] and human [24–27] cells to fully transformed cells with a single on- cogene under specific experimental (significant overexpression of oncogene) and culture con- ditions. Culture conditions significantly affect proliferative (before senescence) [5] and trans- formation potential of cells [8]. For example, wild type MEFs grown in serum-free medium supplemented with defined growth components (EGF, PDGF, insulin, high density lipoprotein, fibronectin, and transferrin) were refractory to transformation by oncogenic RAS + E1A [7]. Moreover, RAS + E1A-induced chromosome in- stability, colony formation and tumorigenesis of the p53 –/– serum free-MEFs also could be at- tenuated by treating the cells with the free-radical scavenger N-acetylcysteine [7]. Finally, humans live, on average, 30–50 times longer than mice and undergo about 105 more cell divisions in a lifetime (1016 versus 1011 mi- toses) [4]. Nevertheless, epidemiological studies have revealed that the life-time risk of develop- ing cancer is comparable in both species. About 30 % of laboratory rodents have cancer by the end of their 2–3 year life-span and about 30 % of people have cancer by the end of their 70–80 year life-span [4]. Thus, it seems that in vitro (and likely in vivo) transformation process may be fundamentally similar in rodent and human cells and be sig- nificantly affected by non-physiological culture conditions in vitro. Senescence. In contrast to germ cells and cer- tain stem cells somatic cells have a limited lifes- pan, gradually slow in growth, and stop dividing, a process known as replicative senescence [28]. The finite replicative life span of normal cells in culture was first described approximately 50 years ago by Leonard Hayflick [29], and is often termed as the «Hayflick limit» [30]. The precise number of rep- licative doublings exhibited by cultured cells before they reach senescence depends on the species from which the cells are derived, the tissue of origin, and the age of the donor organism [31]. Cultured human primary fibroblastic cells generally display 50 to 80 population doublings (PD) [7, 32, 33], whereas explanted MEFs can divide just for 15–30 PD before undergoing senescence [5, 7]. Primary normal human astrocytes perform only about 20 PD before reaching senescence [34]. Human ke- ratinocytes have an in vitro life span of 15–20 PD in serum-free chemically defined media, whereas keratinocytes grown on feeder fibroblasts proliferate for up to 50 PD [7, 32] and in F medium on feeder fibroblasts for up to 80 PD before senescencing [35]. Most published reports on cultured human epithelial cells have shown active growth for only 10 to 30 PD [32]. Significantly, simple changes in the culture conditions (defined growth factors in- stead of serum) could permit active growth of hu- man mammary epithelial cells for up to 60 PD, whereas addition of oxytocin (endogenous antioxi- dant) gave about 20 PD of increased proliferation [32]. MEFs proliferate for more than 60 PD with no signs of replicative senescence under physiologi- cal oxygen levels (3 % versus 21 %) [7, 8]. Thus, primary cells undergo stress-associated senescence due to in vitro non-physiological standard cultur- ing conditions, including disruption of cell-cell contacts, lack of heterotypic interactions between different cell types, the medium-to-cell ratio, per- sistent signaling pathways activation by mitogens, absence of appropriate survival factors, hyperoxia, and plating on plastic [5]. 38 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan The process of senescence occurs both in vitro and in vivo. Cellular senescence in vivo is now recognized to play an active role as a tu- mor suppressor pathway [36, 37], in the loss of regenerative potential in aging tissues and in the pathogenesis of cardiovascular diseases [38]. Se- nescence in vitro is marked by the appearance of large, flattened vacuolated cells and character- ized by the inability of cells to proliferate despite the presence of a steady supply of abundant nu- trients, mitogens [39], ample room for expansion [33], and by the maintenance of cell viability/ resistance to apoptosis and metabolic activity for months [37, 38, 40]. Once senescence is triggered, cells are not capable of re-entering the cell cycle or developing into tumors [36]. Moreover, senes- cent cells secrete a plethora of factors primarily involved in insulin-like growth factor and trans- forming growth factor signaling, extracellular matrix remodeling, and inflammation. Altogether these secreted factors were referred to as the «Se- nescence-Messaging Secretome» or the «Senes- cence-Associated Secretory Phenotype» [33]. Senescent cells can be distinguished from pre- senescent, immortal, quiescent or terminally dif- ferentiated cells by histochemical detection of the biomarker senescence-associated -galactosidase [41]. Senescence accompanies changes in nuclear morphology and formation of a distinct chro- matin structure, called senescence-associated heterochromatic foci (SAHF). These foci are characterized by the accumulation of histone H3 trimethylated at lysine 9 and recruit heterochro- matin proteins to the genes that are to be stably repressed during senescence [41]. Importantly, formation of SAHF and silencing of genes re- quire an intact pRB pathway, since inhibition of p16INK4A prevents SAHF formation and leads to DNA replication [33, 41]. The onset of senescence is partly attributable to the shortening of telomeres by approximately 50–200 base pairs with each cell division to a threshold where it is recognized as DNA dam- age and thus initiates replicative senescence [31, 33, 38]. Critical telomere shortening and even- tual dysfunction triggers a classical DNA dam- age response involving a number of cellular pro- teins, including ataxia telangiectasia mutated protein (ATM), check point kinase 1/2 (CHK1 and CHK2), p16INK4A, p53, 53 binding protein 1 (53BP1), p21CIP1, nijmegan breakage syndrome 1 protein (NBS1), plasminogen activator protein 1 (PAI1), and phosphorylated histone -H2A.X [41]. These cellular factors cooperate to initiate senescence, thereby preventing cellular prolifera- tion in the presence of damaged chromosomes and hence limiting the acquisition of potential pathogenic mutations [33, 41]. However, telomere attrition is not the only stimulus for replicative senescence. Oxidative stress can induce or accelerate the onset, a phe- nomenon referred to as stress-induced replicative senescence. It occurs in several ways (reviewed in [38]): oxidative stress can activate critical cell cycle tumor suppressor proteins p53 and pRB by oxidative-stress-induced DNA damage such as double strand breaks. Oxidative stress can result in oxidative modifications of triple guanine re- peats (TTAGGG) in sequences of telomeric ends making them more susceptible to breaks and en- hancing the rate of telomere attrition. Oxidative- stress-induced premature senescence might be a function of a direct suppression of telomerase activity. hTERT gene (encodes catalytic subunit of human telomerase) expression is regulated by many transcription factors, including AP1, SP1 and NF- B, all of which are redox regulated [42]. In any case, oxidative stress results in the loss of chromosomal integrity as manifested by chromosomal fusions, recombination and deg- radation, and contributes to DNA damage re- sponses that eventually lead to the irreversible cell-cycle arrest/senescence or cell death through the activation of p53- and pRB-dependent func- tions [24, 43]. Immortalization. Senescence (telomere erosion- induced, oncogene-induced [33], or stress-in- duced [44]) forms a barrier against tumorigen- esis. Overcoming of senescence and acquisition of immortality is an essential step in the pro- cess of malignant transformation [33, 45]. Cel- lular immortalization allows a cell to indefi- nitely proliferate while accumulating genetic abnormalities [27, 46]. Immortalized cells can- not grow over one another (contact inhibition) [47, 48], their proliferation ability is growth fac- tor dependent [49] and their growth is anchor- age-dependent (cells do not form colonies in soft agar) [50–52]. There are several mutually complementary mechanisms that contribute to 39ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells a cell being able to escape senescence and be- come immortal, including telomere length sta- bilization, epigenetic gene silencing by selective promoter methylation, oxidative DNA damage, inactivation of cell cycle regulatory genes such as p16INK4A, p53, pRB, p21CIP1, overexpression of a cellular oncogenic proteins such as c-MYC, BMI1 or through expression of viral oncogenes [41]. In vitro immortalization of various cell types was successfully performed by the introduction of viral genomes/oncogenes (Table 1), telomerase catalytic subunit (hTERT) (Table 2) as well as by enforced expression of transcription factors (e.g. c-MYC, BMI1, ZNF217, or -catenin). Genomes of viruses encode a number of regu- latory and structural proteins but «immortalizating effect» can be attributed only to several of them. For SV40 viral oncoproteins responsible for im- mortalization correspond to the portion of the viral chromosome expressed early after infection, which encodes two proteins, the large T-antigen and the small t-antigen [92, 93]. For the adenovi- ruses viral oncoproteins are encoded by a subset of the early genes and termed the E1A proteins and the E1B proteins [93]. Experiments with the hu- man papilloma viruses uncovered a similar set of early proteins called the E6 and E7 proteins [93, 94]. In the cell these oncoproteins bind to pRB and p53 causing their ubiquitin-dependent pro- teasomal degradation. It allows going through the cell cycle checkpoints in an uncontrolled manner. Role of pRB and p53 signaling pathways in cell cycle regulation is presented in Figure. Cells ex- pressing these viral oncogenes continue proliferat- ing beyond the population doubling level, at which their untreated counterparts become senescent, but they eventually cease proliferating in a state referred to as crisis [95–97]. A small number of cells within the population may acquire the ability to escape from crisis and form an immortalized cell line. In all such cell lines examined, escape from crisis has been shown to be associated with activation of a telomere maintenance mechanism. Viral oncoproteins can bind to multiple other cellular proteins [92, 98], including several tran- scription factor complexes involved in hTERT transcription regulation [94]. For example, hu- man papilloma virus E6 protein, via direct bind- ing, increases c-MYC efficiency in activating the hTERT promoter and, on the other hand, E6 is able, through its association with E6AP, to promote the degradation of the hTERT promoter transcriptional repressor NFX1-91 [94]. Usage of both viral oncogene and hTERT to induce immor- talization has also been reported. For instance, pre-adipocytes, bone marrow stromal cells and ovarian surface epithelial cells were immortal- ized by introduction of HPV E7 and hTERT [96]. Mechanistically, thus, process of immortalization induced by viruses corresponds to a process of cell cycle checkpoint proteins inactivation (pRB and/ or p53) and restoration of telomerase activity re- sulting in telomere ends stabilization. Ectopic expression of hTERT alone in pre- senescent or still dividing cells can effectively Schematic representation of pRB and p53 signaling pathways. p16INK4A inhibits CDK4 and CDK6 preventing interaction with D-cyclins. CDK4 and CDK6 phosphorylate pRB leading to a partial loss of its ability to repress the E2F. When pRB-E2F suppressive interaction is relaxed, E2F transactivates genes involved in G1/S transition and in the initiation of DNA replication in S phase. CDK2-cyclin E complexes can further phosphorylate pRB resulting to complete its release from interacting with E2F and, thus, promoting S phase progression. P14ARF/p19ARF is an antagonist for MDM2 which, in turn, regulates p53 stability through its ubiquitin ligase activity. ARF sequesters MDM2 resulting in p53 activation and stabilization. p53 induces p21CIP1 expression. p21CIP1 associates with cyclin D-CDK4/6, E/A-CDK2, and cyclin B-CDK1 complexes, and has a universal inhibitory activity towards these CDKs thereby regulating G1/S transition, S and M phases of cell cycle 40 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan immortalize them (Table 2). The native hTERT locus is embedded in a large nuclease-resistant chromatin domain in most normal human cells [97]. As a result, the hTERT promoter is strin- gently repressed in somatic primary cells. But in immortalized and tumor cell lines hTERT is of- ten up-regulated, and these cells are capable to maintain stable telomere lengths by activation of a telomere maintenance mechanism; also, there is non-telomerase alternative mechanism of telo- mere maintenance [95–100]. Except cases with ectopic hTERT expression, in otherwise immor- talized (and tumor) cells hTERT gene activa- tion can occur in several ways probably mutu- ally complementary to each other: through gene amplification; nonreciprocal translocation by chromosomal breakage at the hTERT locus and subsequent ligation to heterologous sequences by non-homologous end joining (NHEJ) mecha- nisms resulting in the chromosomal rearrange- ments upstream of its promoter; the activation of c-MYC and inhibition of histone deacetylases (HDACs) (reviewed in [97]). Hyperoxia or ad- dition of exogenous H2O2 was shown to induce senescence of fibroblasts despite hTERT overex- pression and exogenous H2O2 prevented hTERT- dependent immortalization of normal endothe- lial cells, whereas N-acetylcystein (antioxidant) permitted hTERT-dependent immortalization of endothelial cells [38]. Indeed, oxidative stress re- gulates hTERT at many levels, such as its gene ex- pression, activity, and sub-cellular localization [42]. Immortalization of human and rodent cells was also achieved by different transcription factors. The c-MYC protein is a basic helix-loop-helix leucine zipper transcription factor that modulates Table 1 Immortalization of human cells by viral genomes/oncogenes Indications. SV40 – simian polyomavirus; HPV16/18 – human papilloma virus types 16 and 18; Ad5 and 12 – adenovirus type 5 and 12. Cell type Immortalizing agent Ref. Astrocytes HS74BM diploid fetal bone marrow fibroblasts IMR-90 diploid lung fibroblasts Ciliary epithelial cells Fetal liver epithelial cell Mammary epithelial cells Prostate epithelial cells Tracheal epithelial cells Uroepithelia cells Cervical epithelial cells Epidermal keratinocytes Epidermal keratinocytes Esophageal epithelial cells Foreskin keratinocytes Gingival keratinocytes HFE keratinocytes Mammary epithelial cells Mammary epithelial cells Uroepithelial cells (from ureteral uroepithelium) Urothelial cells (from ureteric or bladder tissue) WHE-7 fetal fibroblast Embrionic kidney cells HEK293 Bronchial epithelial cells Epidermal keratinocytes SV40 T antigen SV40 SV40 SV40 SV40 T antigen SV40 SV40 SV40 SV40 HPV16/18 HPV16 HPV16 E6+E7 HPV16 E6/E7 HPV16/18/ 31/ 33 HPV16 E6 HPV16 E7 HPV16 HPV16 E6+E7 HPV16 E7 HPV16 E6+E7 HPV16 E6 Ad5 Ad12-SV40 fusion genome Ad12-SV40 fusion genome [53] [54] [55, 56] [57] [58] [59] [60] [61] [62] [63, 64] [65, 66] [67, 68] [69] [70, 71] [72] [73] [74] [75] [76] [77] [72] [78] [79] [80] 41ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells Table 2 Molecular characteristics of immortalized human cell lines Indications. RPE-340 – retinal pigment epithelial-340 cells; MDAH 087 – skin fibroblasts derived from a patient with Li-Fraumeni syndrome; Y-27632 – Rho kinase (ROCK) inhibitor; 4-NQO 4-nitroquinoline 1-ox- ide; ALT – alternative telomere maintenance mechanism; « » – overexpressed; « » – downregulated; «+» – unchanged level; * for hTERT – overexpressed; «–» – undetected; N/A – not analyzed. Cells Immort. agent p16INK4A pRB p53 p21CIPI hTERT* Ref. Adenoid epithelial cells and foreskin keratinocytes Ameloblastoma cells BJ fibroblasts, RPE-340 cells BJ fibroblasts Cen3 fibroblasts Esophageal epithe- lial cells Foreskin fibroblasts Gingival and perio- dontal ligament fibroblasts Dermal keratino- cytes Mammary epithe- lial cells Epi gingival kera- tinocytes Esophageal epithe- lial cells Mammary epithe- lial cells WHE-7 fetal fibro- blasts Foreskin fibro- blasts Prostate epithe- lial cells Mammary epithe- lial cells Oral keratinocytes Foreskin keratino- cytes KMST-6 fibroblasts MDAH 087 skin fibroblasts OUMS-24F fibro- blasts hTERT hTERT hTERT hTERT hTERT hTERT hTERT hTERT hTERT hTERT HPV E6 HPV16 E6/E7 HPV16 E6 + E7 HPV16 E6 c-MYC c-MYC ZNF217 Cyclin D1 + domi- nant-negative p53 Y-27632 60Co Aflatoxin B1/X-rays 4-NQO – – +/– N/A / – deletion – – – – N/A – + – N/A N/A + – – + + hyper phosphorylated N/A + + unaffected N/A + unaffected + hyper phosphorylated + hyper phosphorylated N/A E2F1 elevated + hyper phosphorylated + hyper phosphorylated N/A N/A + unaffected N/A N/A + hyper phosphorylated + hyper phosphorylated + hyper phosphorylated + + + + + mutated + +/ mutated N/A + + N/A N/A + + + + mutated + N/A + mutated N/A + + N/A N/A +/– N/A N/A + + + N/A N/A N/A + + + + + + + + + + + + N/A N/A N/A + + ALT + ALT ALT ALT [81] [82] [83] [84] [24] [69] [85] [72] [86] [87] [72] [69] [75] [72] [88] [89] [90] [91] [28] [72] [72] [72] 42 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan expression of a cohort of genes, including those that function to promote cell growth and cell cy- cle entry [88, 101]. c-MYC up-regulates certain cyclin-dependent kinases (CDK4) and cyclins (A, B1, D1, D2, and E), and represses cycline- dependent kinase inhibitors (p15INK4B, p21CIP1, and p27KIP1) [48, 101, 102]. Mechanisms can be direct or indirect: for example, c-MYC directly binds to the cyclin B1 promoter, but optimal in- duction of expression occurs only when p53 is concurrently inactivated [102], whereas cyclin D1 expression is positively regulated through MYC/ miR-378/TOB2/cyclin D1 functional module in human mammary epithelial cells [103]. p21CIP1 expression is regulated both negatively and posi- tively by c-MYC [104]. Induction of p21CIP1 by c-MYC overexpression was p53-dependent in nor- mal human and mouse fibroblasts and was associ- ated with G2 arrest, whereas, inversely, c-MYC repressed p21CIP1 transcription in p53-null mouse cells and in a human adenocarcinoma cell line [105]. Moreover, the hTERT promoter contains the MYC binding site (E-box) and is a direct tran- scriptional target of c-MYC [106]. c-MYC expres- sion was reported to result in successful immor- talization of rat kidney cells [22], mouse neural precursor cells [107], human neural stem cells (by v-MYC and c-MYC T58A mutant) [49], prostate epithelial cells [89], and foreskin fibroblast cells [88]. Interestingly, foreskin fibroblast cells had in- creased levels of p16INK4A and p53 and functional both p16INK4A–pRB (pRB phosphorylation was reduced) and p53–p21CIP1 parthways. Prostate epithelial cells preserved functional p53-p21CIP1 pathway and had elevated p16INK4A but, never- theless, pRB phosphorylation was maintained. Moreover, c-MYC allowed to tolerate ectopically overexpressed p16INK4A in prostate epithelial cells, whereas p16INK4A overexpression in foreskin fibro- blast resulted in senescence. Foreskin fibroblast cells also showed epigenetically silenced p14ARF (unfortunately, p14ARF status was not analysed in prostate epithelial cells). Rodent cells immortal- ized by c-MYC characteristically inactivate the ARF–p53–p21CIP1 pathway by loss of either func- tional p53 or 19ARF [88]. p14ARF/p19ARF is unique among c-MYC regulators. It selectively inactivates the hyperproliferative and transforming func- tions of c-MYC without affecting normal cell cycle progression or preventing c-MYC-mediated apoptosis [108]. Thus, p53 or p14ARF/p19ARF in- activation is likely beneficial in cells immortalized by c-MYC. Other transcription factor BMI1, a member of the Polycomb group of transcriptional repres- sors [109], was initially identified as an oncogene that cooperates with c-MYC in lymphomagenesis [110]. Moreover, BMI1 is positively regulated by c-MYC [37]. It was reported that overexpression of BMI1 down-regulated p16INK4A and p19ARF expression in mouse embryonic fibroblasts and resulted in their immortalization [96], immortal- ized primary human mammary epithelial cells (HMEC) [111] and nasopharyngeal cells [112]. In both latter cases immortalization was accom- panied by telomerase activation. BMI1 caused the bypass of replicative senescence in normal human oral keratinocytes but did not immortal- ize them (no hTERT activation) [113]. BMI1 introduction along with human papilloma virus E6 gene but not with E7 immortalized oral ke- ratinocytes and it was associated with telomerase activation [113]. Introduction of BMI1 as well as p16INK4A-specific short hairpin RNA into hu- man epithelial cells derived from skin, mammary gland and lung suppressed p16INK4A expression and extended cells life span; subsequent intro- duction of hTERT in these cells resulted in their efficient immortalization with following mainte- nance of near normal diploidy [96]. The reason why some cell types become immortalized after BMI1 introduction alone, whereas other cells do not is unclear. It was speculated that BMI1-in- duced immortalization mechanism may be tissue- dependent or because the cultured cells already underwent critical steps towards immortalization [113]. The mechanism whereby BMI1 promotes evasion of senescence involves, among other tar- gets, transcriptional silencing of CDKN2A (cy- clin-dependent kinase inhibitor 2A, which en- codes both p16INK4A and p14ARF) and CDKN2B, which encodes p15INK4B [42, 110, 114]. Transcription factor ZNF217 was able to im- mortalize human primary HMECs disturbing ARF-p53-p21CIP1 pathway [90]. -Catenin, a member of the Armadillo (ARM) repeat protein superfamily, activates transcription of target genes primarily by associating with the T cell factor/lym- phoid enhancer-binding factor (TCF/LEF) family [115, 116]. Expression of -catenin immortalized 43ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells primary mouse melanocytes directly repressing the expression of p16INK4A by binding to its promoter [117]. Other well known targets of -catenin are c-MYC and cyclin D1 [115]. In fact, all mentioned targets of discussed tran- scription factors are only the «top of an iceberg». It was established that c-MYC regulates a total of 1469 target genes in HeLa cells and human primary fibroblasts [118], -catenin in HCT116 colorectal carcinoma cells directly bound in vivo to more than 400 target genes [115]. ZNF217 was shown to target 103 genes in breast cancer cell line MCF7, 44 genes in colon cancer cell line SW480, and 101 genes in teratocarcinoma cell line Ntera2 [119]. Moreover, NF- B, STAT3, ER , JUN, ELK4, CEBP, and ETS1 were found among transcrip- tional regulator genes up-regulated by c-MYC in human B cell line P493 [120], and EPAS1, ERF, FHL2, JUN, MNT, MYT1, RPO1-2, SOX4, TEAD4, TIEG1, and ZFP28 in pancreatic -cells [121], which, in turn, regulate additional gene cohorts resulting eventually in global change in gene expression. C-MYC can regulate overall up to 10–15 % of all genes [120, 122], among which there are those regulating replication and repara- tion in S phase and chromosome separation dur- ing M phase [121–124]. Spontaneously immortalized cells emerge at an extremely low frequency (about 10–7) during crisis in vitro [85, 97], but show the same gen- eral changes in cell cycle checkpoint pathways as all otherwise immortalized cells [125–127]. Thus, overwhelming majority of immortalized cells irrespective of «immortalizing agent» do not express p16INK4A cell cycle suppressor and this correlates with pRB hyperphosphorylation (inac- tivation) (Table 2). Indeed, it has been estimated that more than 70% of human immortalized and cancer cell lines lack functional p16INK4A due to promoter methylation, mutation, or homozygous deletion. In many instances the deletions affect both p16INK4A and p14ARF/p19ARF, but a substan- tial proportion of the missense mutations exclu- sively affect p16INK4A, suggesting that p16INK4A itself plays significant and non-redundant role in tumor suppression [128]. Spontaneous reduction in p16INK4A expression due to promoter methyla- tion (most often) or otherwise mechanisms during in vitro propagation of normal primary cells has been documented, for example, in HMEC [96, 129], fibroblasts derived from lung [96], human keratinocytes [96, 130], and human astrocytes [34]. Molecular mechanism of p16INK4A gene in- activation by epigenetic deregulating methylation during progression from primary cells to immor- talized and pre-malignant cells is complex; it is under intensive investigation and may be differ- ent in mouse and human cells [128, 131–137]. Nevertheless, in contrast to frequent loss of p16INK4A expression in vitro, another well doc- umented fact is that p16INK4A is overexpressed in certain samples of different cancer types. In tumors increased p16INK4A expression correlates statistically with RB loss of heterozygosity [138]. In spite of being tumor suppressor, overexpres- sion of p16INK4A, nevertheless, correlates with a poor prognosis and seems to be an unfavorable prognostic indicator [138]. ARF–p53–p21CIP1 pathway is likely less critical for immortalization of human cells than p16INK4A–pRB, because approximately in a half of analyzed works it was apparently functional (Table 2). Moreover, Odell et al. [139] exam- ined more than a hundred spontaneously im- mortalized MEF cell lines and found that at least half of them had neither a p53 mutation nor loss of p19ARF. Nevertheless, it is neces- sary to take into account that analysis of ARF- p53-p21CIP1 pathway in most works (where it was shown functional) was performed once in certain population doubling (PD), but it could be inactivated later. For instance, hTERT im- mortalized cen3tel fibroblasts up to 108 PD had wild-type p53 sequence, whereas at late PDs (165 and 366 PDs) had a mutation in codon 161 [24]. Thus, it is possible to conclude that for suc- cessful immortalization cells must overcome se- nescence by inactivating p16INK4A–pRB and /or ARF-p53-p21CIP1 and crisis by maintaining their telomeres by activation of hTERT expression or by an alternative mechanism for lengthening telomeres (ALT) [99, 100]. However, it needs to keep in mind that every oncogene/«immortalizing agent» introduced into a cell has a great number of targets and multidirectional effects rather than being one-way agent. Thus, the process of im- mortalization is not simply a number of well de- fined events like inactivation of cell cycle nega- tive regulators and activation of telomerase but, instead, is associated with karyotype/genome ab- 44 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan normalities (aneuploidy/gain or loss of additional chromosomes, translocations, deletions and am- plifications) and, as a consequence, with global changes in gene expression [165]. All immortal- ized cells have abnormal karyotypes irrespectively of «immortalizing agent» (Table 3). Also, significant changes in global gene ex- pression can be reached through aberrant meth- ylation of promoters. In HMECs during im- mortalization global aberrant DNA methylation changes occured in a stepwise fashion [129]. The first aberrant DNA methylation step coincided with overcoming stasis, and resulted in few to hundreds of changes, depending on how stasis was overcome (stress-inducing serum-free me- dium, benzo(a)pyrene or p16INK4A shRNA). A second step coincided with crisis/immortaliza- tion and resulted in hundreds of additional DNA methylation changes regardless of the immortal- ization pathway [129]. All together, it explains why across cell types and model systems genes in the cell cycle path- way, cytoskeletal genes, IFN pathway, IGF path- way, MAP kinase pathway, and oxidative stress pathway were identified as regulators of senes- cence/immortalization [41, 166]. It is worth no- ticing that virus oncoproteins induce more pro- found karyotype changes because of simultaneous ablation of pRB and p53 pathways, inactivation of which is directly linked with aneuploidy/poly- ploidy. In contrast, hTERT alone immortalized cells are suggested to be apparently genetically stable frequently showing near diploid karyotypes with lower abnormalities than otherwise immor- talized cells [47, 142, 143, 147, 167–171]. It is clear that telomerase introduction into a cell can Table 3 Karyotype abnormalities in immortalized cells Immortalizing agent Ref. Immortalizing agent Ref. Cells immortalized by hTERT Human adenoid epithelial cells and foreskin keratinocytes Human fibroblasts from two centenarian individuals Human normal fibroblasts Sheep fibroblasts Human bone marrow endothelial cells Human small airway epithelial cells Human mammary epithelial cells Human myometrial and uterine leiomyoma cells Swine umbilical vein endothelial cells Human mesenchymal stem cells Human fetal hepatocytes Human meibomian gland epithelial cells Cells immortalized by SV40 T large antigen Rabbit kidney epithelial cells Human corneal epithelial cells Human gingival keratinocytes Human nasopharyngeal epithelial cells Human bronchial epithelial cells Human mammary epithelial cells [81] [140] [83, 84, 141, 142] [143] [144] [145] [46] [146] [47] [147] [148] [149] [150] [151] [152] [153] [154] [155] Cells immortalized by HPV16/18 E6/E7 Human epidermal keratinocytes Human smooth muscle cells Human nasopharyngeal epithelial cells Human extravillous cytotrophoblasts Human bronchial epithelial cells Spontaneously immortalized cells MEFs Syrian hamster embryo cells Human epidermal cells Human keratinocytes Murine neural crest-derived corneal progenitor cells Immortalized by otherwise ways Mouse embryos cells by v-SIS or K51 oncogenes Human mesenchymal stem cells by E6/E7 plus hTERT or BMI1, E6 plus hTERT Human meibomian gland epithelial cells by SV40 LT antigen plus hTERT Human bronchial epithelial cells by hTERT plus CDK4 or HPV16 E6/E7 Human bronchial epithelial cells by BMI1 plus hTERT Human prostate epithelial cells by c-MYC Oral keratinocytes by cyclin D1 plus mutant p53 [156] [157] [153] [158] [159] [160] [161] [125] [162] [126] [163] [147] [149] [159] [164] [89] [91] 45ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells stabilize telomeres preventing numerous chromo- somal aberrations occuring via telomere dysfunc- tion and the breakage-fusion-bridge mechanism [165]. Nevertheless, although BMI1 and hTERT immortalized human embryonic stem cells at passage 40 [172], as well as hTERT immortalized human neural progenitor cells isolated from the ventral telencephalons of first trimester embryos after more than 40 passages (80–120 PD) [170] had normal karyotypes, in contrast, hTERT im- mortalized bone marrow endothelial cell clones showed numerous abnormalities after 75 PD (more than 160 days) [144]. Interestingly, mass culture of these cells at 65 PD (120 days) had 47 chromosomes without any structural abnormali- ties and served this karyotype at 135 PD [144]. Also, prolonged cultivation of telomerase-immor- talized human fibroblasts led to a premalignant phenotype, although hTERT-immortalized cells behaved similarly to primary cells during the first 150 PDs [141]. The possible pitfall of «normal» or near diploid karyotypes of hTERT immortal- ized cells can result from exploiting conventional cytogenetic techniques for karyotyping, which do not allow detecting the subchromosomal aberra- tions. In contrast, for example, SNP and CGH arrays revealed multiple genomic abnormalities in tumors with near diploid katyotypes. How do immortalized and tumor cells become aneuploid? p53, a well known «genome safeguard», plays multiple roles in maintaining genomic stability in somatic cells. Loss of p53 functions promotes on- cogenesis by inducing chromosomal instability and aneuploidy [173-176] and enabling efficient accu- mulation of genetic mutations [177]. Loss or muta- tional inactivation of p53 results in a high frequency of centrosome amplification in part via allowing the activation of CDK2-cyclin E (as well as CDK2- cyclin A), which is a critical factor for the initiation of centrosome duplication [178]. It also allows im- mature escaping from cell cycle G2 checkpoint ar- rest through inability of p21CIP1 to inactivate CDK2, and this leads to reinforcement of CDK2-dependent NF-Y phosphorylation and NF-Y dependent tran- scription of the cell cycle G2-regulatory genes, in- cluding CDK1, CDC25, cyclin A and B [179]. There is also a link between pRB inactiva- tion, cell aneuploidy and chromosome instabil- ity (CIN). Aneuploidy and CIN results from persistent defects in mitotic fidelity, and several mechanisms have been described that cause cells to missegregate whole chromosomes [176, 180, 181]. More than 50 proteins are able to trigger polyploidy/aneuploidy when are appropriately misregulated (mutation, depletion, knockdown or overexpression) [182]. If the dosage of any one of many proteins involved in ensuring chro- mosome segregation fidelity is disrupted by the missegregation of the chromosome carrying that gene, the resulting imbalance can further com- promise chromosome segregation accuracy [176]. Importantly, pRB-E2Fs pathway directly regu- lates genes involved in bipolar spindle formation, chromosome-spindle association, chromosome cohesion, and the spindle assembly checkpoint (SAC) [183]. Acute pRB suppression in IMR90 cells [184], HCT116 cells [185], primary hu- man fibroblasts [186], mouse embryonic fibro- blasts [180], and mouse adult fibroblasts [187] caused misregulation of chechpoint genes [180, 185, 186] and, as a consequence, gave rise to centrosome amplification, multipolar spindles, anaphase bridges, lagging chromosomes, and mi- cronuclei harbouring whole chromosomes result- ing in polyploidy/aneuploidy and cancerogenesis [180, 184–188]. Moreover, pRB influences mi- totic chromosome condensation in E2F-inde- pendent manner, and loss of pRB function can influence chromosome loss irrespectively of pro- liferation [189]. pRB can interact with the con- densin II subunit CAP-D3, and this interaction is necessary for chromosome compaction in mitosis [189]. pRB depletion compromises centromeric localization of CAP-D3/condensin II and chro- mosome cohesion, leading to an increase in in- tercentromeric distance and deformation of cen- tromeric structure [181]. These defects promotes merotelic attachment (occurs when one kineto- chore is attached to both mitotic spindle poles), resulting in failure of chromosome congression and an increased propensity for lagging chromo- somes following mitotic delay [181]. In contrast to pRB, p107 and p130 (also members of retinoblastoma family) are rarely found inactivated in human tumors [190], and this fact determined predominant research on pRB, but it hardly proves the unique importance of pRB in cell cycle regulation. Equally weighty roles of p107 and p130 may well be masked by a functional redundancy that they have with one 46 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan another. Such redundancy would drastically re- duce the likelihood of their elimination from tu- mor cell genomes during tumor progression [191]. Indeed, these proteins are part of a «tumor-sur- veillance» mechanism and can suppress tumori- genesis [190, 192–194]. Another consequence of pRB and p53 path- ways deregulation is compromised DNA damage surveillance and repair. More than 70 genes have been identified that have roles in DNA damage surveillance and repair [195], and many of them are regulated by pRB and p53 [183, 195]. pRB and/or p53 pathways deregulation and hTERT expression (or ALT) are markers and indis- pensable conditions of immortalization. Deregu- lated pRB and/or p53 pathways inevitably leads to numerical and structural chromosome aber- rations. A cell attains immortality by a global change in gene expression, which accompanies karyotype changes. Although a few common chro- mosome aberrations might have been observed in different immortalized cell, karyotype changes, in general, have stochastic nature. Immortalized cells with aberrant karyotypes are prone to malig- nant transformation. Transformation. Cancer cells display several hallmarks that can be distinguished from those of normal counterparts. These include immortaliza- tion (bypass of senescence), evasion of apoptosis, immune destruction and anti-growth signals, growth factor independence, reprogramming of ener- gy metabolism (enhanced glycolysis), anchorage- independence, resistance to contact inhibition, mi- gration, invasion/degradation of matrix components, angiogenesis, metastasis, inflammation, and geno- me instability, which generates the genetic diver- sity accelerating acquisition of all listed hallmarks [31, 196]. In addition to cancer cells, tumors ex- hibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the «tumor microenvironment» [196]. Cancer genes are often classified according to whether they function in a dominant or recessive manner at the level of the cancer cell. Dominant cancer genes (also known as oncogenes) require only one of the two parental alleles present in a normal cell to be mutated, and the encoded protein is usually constitutively activated by the mutations. Recessive cancer genes (also known as tumor suppressor genes) require mutation of both parental alleles, and these usually result in inactivation of the encoded protein. More than 80 % of the currently known cancer genes are dominantly acting [3]. Census of cancer genes lists 467 genes (data on December 2011, www. sanger.ac.uk/genetics/CGP/Census), which are supposed to be causally implicated in cancer de- velopment when appropriately changed (point mutations, deletions, translocations or ampli- fications) [197]. However, studies in mice have suggested that more than 3000 genes, when ap- propriately altered, may have the potential to contribute to cancer development (see reference in [198]). The process of cellular transformation has been intensively studied in vitro using cell-culture tech- niques. Most research works on this issue satisfy four criteria: the cells are immortalized, i.e., can grow indefinitely in culture; the cells can efficiently form colonies in soft agar; the cells can develop tumor in immunodeficient mice; the xenograft or orthotopic tumor in the mouse shows malignant histology to exclude a pseudo-tumor or a benign tumor [267]. Actually, there are a few interesting outliers. For example, cells obtained by stable overexpression of cyclooxygenase 1 in spontaneously immortalized human umbilical vein endothelial cells underwent contact inhibition, failed to grow under anchor- age-independent conditions but grew aggressively as tumors in mice [202]. Human primary foreskin fibroblasts in which E1A+H-RASV12+MDM2 were introduced, although able to form colonies in soft agar and tumors in nude mice, were not immor- tal and, if maintained in culture for an extended period of time (40–50 generations), underwent a crisis phase characterized by dramatically reduced proliferation and adoption of a senescent pheno- type [10]. Cells were telomerase-negative, only few of them eventually survived this phase and these cells became telomerase-positive [10]. MAPK and PI3K-AKT signaling pathways in transformation: a double-edged sword. One prom- inent hallmark of transformed in vitro cells irre- spectively of transforming agent is upregulation of RAS-dependent extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK) pathway, phosphoinosit- ide-3-kinase (PI3K)-mammalian target of ra- pamycin (mTOR)-AKT pathway and overex- 47ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells pression of CDKs/cyclins. Actually, besides MAPK and PI3K/AKT signaling, other path- ways can also be activated. Nevertheless, RAS- dependent extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase [268–270] and phosphoinositide-3-ki- nase (PI3K)-mammalian target of rapamycin (mTOR)-AKT cascades [271] are the key signal transduction pathways responsible for integrat- ing the different environmental signals and re- laying the information to the cell cycle control system. Both these pathways are hyperactivated frequently in transformed cells in vitro and tu- mors in vivo, and are involved in regulation of all aspects of normal and tumor cell biology (e.g., cell growth, proliferation, apoptosis, migration, invasion etc). As it is reviewed in [268-270], ERK1/2 are required for cyclin D1 expression via regulation of FOS family members and c-MYC transcrip- tion factors, as well as inhibition of TOB1 and JUND, cyclin D1 expression negative regula- tors. The ERK pathway may assist in both the assembly and stabilization of cyclin D1-CDK4/6 complexes via HSC70. There are also a few re- ports implicating MAPK pathway in the regu- lation of cyclin D2 and cyclin D3 expression. ERK activity is required for proper nuclear translocation of CDK2, and in the nucleus ERK regulates phosphorylation of a CDK2 activat- ing site. ERK can phosphorylate two of four phosphorylation sites of the cytoplasmic reten- tion sequence of cyclin B1, which are neces- sary for nuclear localization of cyclin B1. ERK futher contributes to CDK1-cyclin B activation via RSK/MYT1/CDK1-cyclin B pathway. The RAS-ERK signaling pathway is involved in the mitogen-induced downregulation of p27KIP1. The degradation of p27KIP1 at the G1/S transition depends on the accumulation of cyclin E and concomitant activation of CDK2, events that are conditional on earlier activation of cyclin D- CDK4/6 complexes by the ERK pathway. Activation of ERK markedly enhances c- MYC protein stability, which can transcription- ally upregulate expression of certain cycline- dependent kinases (CDK4) and cyclins (A, B1, D1, D2 and E), and represses cyclin-dependent kinase inhibitors (p15INK4B, p21CIP1 and p27KIP1) [48, 101,102, 272]. ERK1/2 dislodge pRB from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently pro- moting E2F activation and cell cycle entry [273]. Pyrimidine nucleotides serve as essential precur- sors for the synthesis of RNA and DNA, phos- pholipids, UDP-sugars and glycogen [268, 270]. The rate-limiting step in the pyrimidine pathway is catalysed by the carbamoyl-phosphate synthe- tase enzyme, which is part of the large multifunc- tional protein CAD [268, 270]. ERK2 directly phosphorylates CAD activating it. ERK may im- pact on global protein synthesis through direct regulation of ribosomal gene transcription [268]. Over a hundred putative AKT substrates have been reported. Targets among cell cycle regulating proteins are reviewed in [43, 274, 275]. GSK3- mediated phosphorylation of cyclin D and cy- clin E and the transcripton factors c-JUN and c-MYC, which all play a central role in the G1- to-S phase cell-cycle transition, targets them for proteasomal degradation. Phosphorylation and inhibition of GSK3 by AKT enhances the sta- bility of these proteins. AKT reduces p21CIP1 protein level through downregulation of p53- mediated transcription and activation of MDM2, and inhibits p27KIP1 expression via inactivation of FOXO family of transcription factors. AKT phosphorylates both p21CIP1 and the p27KIP1 cy- clin-dependent kinase inhibitors leading to their cytosolic sequestration and phosphorylates and deactivates pRB leading to the activation of E2F. AKT induces p27KIP1 degradation via GSK3 /c- MYC/p27KIP1. Aberrant activation of mTORC1 is a common molecular event in a variety of cancers [276, 277]. Activation of the AKT and ERK pathways acts in a synergistic manner to promote mTORC1 signaling through phosphorylation of a tuberous sclerosis complex 2 (TSC2), GTPase activator protein (GAP), leading to the disruption of the TSC1-TSC2 complex as an inhibitor of RHEB, which in turn regulates mTORC1. AKT phos- phorylates residues of TSC2 distinct from those phosphorylated by ERK [278]. Furthermore, the kinase RSK, a direct downstream substrate of ERK, can also phosphorylate TSC2 to inhibit the func- tion of TSC1/TSC2 complex [270]. The S6K1 and 4E-BP1/eIF4E pathways represent critical mediators of mTORC1-dependent cell cycle con- trol [279, 280] by promoting the cap-dependent 48 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan translation of many target mRNAs, including those encoding cyclins and c-MYC [274, 281]. mTORC2 also contributes to cell size and cell cycle regulation via AKT activation and, thus, contributing to TSC2 inactivation [275]. Nevetheless, it should be noted that the induc- tion of cell cycle arrest by hyperactivation of the ERK1/2 pathway does occur in some cell lines and is frequently observed in non-immortalized primary cells. Expression of activated forms of RAS, RAF or MEK1 was shown to elicit cell cycle arrest in primary fibroblasts, Schwann cells, hepatocytes, T lymphocytes, keratinocytes, astro- cytes, and epithelial intestinal cells (reviewed in [268]). Notably, the proliferation arrest observed in primary fibroblasts, astrocytes and epithelial intestinal cells is permanent and phenotypi- cally related to cellular senescence [268]. This phenomenon is not restricted to MAPK path- way. AKT overexpression induced senescence of primary and immortalized esophageal epithelial cells [282], primary MEFs [283], primary human aortic endothelial cells, human dermal microvas- cular endothelial cells, and human umbilical vein endothelial cells [284, 285]. Moreover, senes- cence can be triggered in human cells by overex- pression of E2F1/3, CDC6, MOS or deletion of PTEN and NF1 (reviewed in [36]). A robust and prolonged activation of ERK1/2 causes G1 arrest due to long-term p21CIP1 in- duction [286, 287] and CDK2 inhibition and also induces the expression of p53 and the CDK inhibitors p16INK4A and p15INK4B in certain cell lines [36, 268]. Indeed, ERK pathway can induce p21CIP1 transcription [287], translation [288], mRNA stabilization [289] and block proteasome- mediated p21CIP1 degradation [287]. Constitutive activation of AKT promotes senescence-like ar- rest of cell growth via a p53/p21CIP1-dependent pathway and this action is at least partly medi- ated by the forkhead transcription factor [284]. On the other hand, hyperactivated MAPK and PI3K-AKT pathways were documented in most, if not all, tumors and elevated p21CIP1 ex- pression was highlighted, for example, in rectal stromal tumors [290], lung adenocarcinomas [291], bladder tumors [292], colorectal carci- nomas [293], ependymomas and astrocytomas [294], hepatocellular carcinomas [295], choroidal melanoma tumors [296], and rhabdomyosarcoma cells [288]. Importantly, in these cancers p21CIP1 expression was associated with tumor malignancy and poor prognosis but not with long-term sur- vival as it was expected. Thus, oncogene-induced senescence occurs in primary cells, some immortalized and in benign but not in advanced tumors. It suggests that tu- mor cells gain resistance to p21CIP1-mediated scenesence and inhibition of CDK/cyclin com- plexes. Oncogene-induced senescence can be bypassed by inactivating pRB and p53. Accord- ingly, if pRB and/or p53 are inactivated in a cell before an oncogenic event, senescence should be averted what is supported by numerous in vivo mouse modeling studies and by genetic analysis of human tumors [36]. Moreover, RB deficiency sharply increases the ability of RAS to bind gua- nine nucleotides, resulting in its activation [297]. Importantly, p21CIP1 (and p27KIP1 to a lesser degree) functions as an assembly- and activity- promoting factor for cyclin D-CDK4, cyclin E/A-CDK2, and cyclin B-CDK1 complexes when p21CIP1 level is below a certain threshold, after which the presence of excess p21CIP1 be- comes inhibitory. Stoichiometry of p21CIP1 is critical to allow or inhibit kinase activity [104, 286]. When one p21CIP1 molecule is binding to cyclin-CDK, the complex is catalytically active, while binding of several p21CIP1 subunits inhibits the complex. Thus, simultaneous overexpression of CDKs/cyclins with p21CIP1 would create more active complexes fostering cell cycle progression and resistance to antimitotic stimuli. Interestingly, in contrast, loss or decrease of cyclin dependent kinase inhibitor p27KIP1 is com- monly seen in many human cancers as lung, breast and prostate adenocarcinomas, gastrointestinal malignancies, brain tumors, and lymphoprolifera- tive neoplasms [298]. Level of p27KIP1 in epithe- lial cancers correlates with the pathologic tumor grade: high-grade, poorly differentiated tumors showing significantly lower p27KIP1 protein than their well-differentiated counterparts [298]. Thus, selection of tumor cells against p27KIP1 is likely beneficial, and p27KIP1 has less profound role in CDK-cyclin complex assembly than p21CIP1. Karyotype evolution, selection and tumorige- necity. Accoding to the Duesberg's evolutionary chromosomal cancer theory [299–302], «activated oncogenes induce neoplastic transformation by 49ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells inducing random aneuploidy. Aneuploidy destabi- lizes the karyotype by unbalancing teams of proteins that segregate, synthesize and repair chromosomes in proportion to the degree of aneuploidy. Aneuploidy initiates and maintains karyotypic evolutions automatically because of the inherent instability of aneuploidy. Occasionally, rare cancer-causing karyotypes evolve stochastically. These cancer- causing karyotypes are then stabilized against the inherent instability of aneuploidy by selection for transforming function within narrow clonal limits of variation. Flexibility and heterogeneity of cancer karyotypes is the basis for the further, spontaneous evolutions that are known as tumor progression, such as metastasis and drug resistance». Most oncogenes deregulate DNA replication, centrosome amplification and chromosome segregation and lead to formation of DNA double strand breaks and chromosome instability. Indeed, oncogene and carcinogene induced chromosome instability is a driving force of cell immortalization and tumor evolution (Stepanenko and Kavsan, in preparation). For example, activated RAS induces DNA double strand breaks in NIH3T3 fibroblasts within a single cell cycle; other oncogenes, including MYC, cyclin E, MOS, CDC25A, E2F1 and sustained delivery of growth factors have similar effects in various cell types and in animal models (re- viewed in [303]). Importantly, most of experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were per- formed using mouse and human cell lines (Table 4 and 5), which represent already immortalized cells with abnormal karyotypes (poly-/aneuploids with se- vere chromosome rearrangements) and are prone to progress to completely transformed cells under culture conditions. Human embryonic kidney 293 cells (also of- ten referred to as 293 cells, HEK 293, or less precisely HEK cells) is widely used human cell line both for basic molecular studies and as a ve- hicle for the production of recombinant proteines and viruses [304]. Originally named simply «293 cells» («293» designates a number of experiment), they were obtained by exposing human embry- onic kidney cell culture to mechanically sheared fragments of adenovirus type 5 DNA (Ad5) [78]. After transformation the cells subcultured more than 100 times could be considered as an estab- lished/immortalized line and contained 4 to 5 fragments of Ad5 genome [78]. The transform- ing region of the human adenovirus is within the left 11 % of the viral genome encoding E1A and E1B proteins which are necessary and sufficient for mammalian cell transformation by Ads [305]. The integration site of the adenoviral DNA was mapped to chromosome region 19q13.2 [305]. Adenovirus-induced chromosome aberra- tions in human cells are well documented fact [306, 307]. Bylund et al. [304] performed cyto- genetic studies on the 293 cells obtained from different sources. Karyotype analysis (G-banding and spectral karyotyping) showed that 293 cells (from ECACC, Salisbury, UK) cultured for less than ten days prior to harvesting was near trip- loid with 62–70 chromosomes/cell and had lots of chromosomal abnormalities. No additional chromosomal changes were found between 293 cells and 293aged cells (in culture 6 months, more 100 PD). Thus, 293 cells exhibit the cytogenetic stability during culturing. Another work on 293 cell karyotype (cells were obtained from ATCC, Manassas, VA, USA) also showed triploidy of these cells [308] but with only partial overlap in chromosome gains/losses comparing with cells analysed by Bylund et al. [304]. Interestingly, original 293 cells obtained by Graham et al. [78] and tested at passage 8 were near-tetraploid and retained this ploidy at passage 38. On the other hand, it was revealed that 293 cell tumorigenic potential correlated with number of passages, that is, low-passage cells (less 52 passages) could not form tumors in mice in 8 weeks, whereas tumorigenicity reached 100% when the passage had exceeded 65 (2 107 cells per injection; 10 of 10 mice had about 0.5 cm3 in size tumors within 2 weeks, after 4 weeks tumor was as large as 2.0 1.5 1.3 cm3) [309]. Nevertheless, there is no correlation between long-time culturing-induced tumorigenesis of parental 293 cells and karyotype instability [304, 309]. The possible reason why prolonged cultiva- tion drives tumorigenic potential of parental cells might be deduced from investigations with NIH3T3 murine fibroblasts, which were used for transformation assays much more frequently than any other cells (Table 5). NIH3T3 cells were ob- tained in 1962 as spontaneously immortalized cells during long culturing using «3T3 proto- col» [160]. Rubin documented [310–315] that spontaneous transformation of NIH3T3 (also of 50 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan Table 4 Transformation of human cells Indications. HMEC – human mammary epithelial cells; SV7tert cells – derived from angiomyolipoma; FoxM1B – member of the Forkhead box transcription factor; RPMS1 – ORF of Epstein-Barr virus; PTTG1 – pituitary tumor-transforming 1; MutCCK2R – cholecystokinin-2 (CCK2)/gastrin receptor intron 4 retained; ESM1 – endocan, dermatan sulfate proteoglycan; CD74 – major histocompatibility complex, class II invariant chain; HCCR1 – human cervical cancer oncogene 1; PDX1 – pancreatic and duodenal homeobox-factor 1; ROBO1 – a member of round about family of transmembrane receptors. Cell type Immortalizing agent Transforming agent Ref. Astrocytes Barrett's epithelial cells BJ fibroblasts cen3tel fibroblasts Colorectal crypt cells HUVEC Embryonic esophageal epithelial cells FHC fetal colon cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells 293 cells HMEC HMEC HMEC HMEC MCF-10A MCF-12A MCF-10A, 12A MCF-10A MCF-10A MCF-10A MCF-10A MCF-10A Oral epithelial cells Ovarian epithelial cells Prostatic epithelial cells SV7tert cells HPV E6 + E7 + hTERT hTERT Primary normal cells hTERT hTERT + SV40 large T antigen Spontaneous HPV18 E6 + E7 Primary normal cells Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 Ad5 hTERT Primary normal cells hTERT Primary normal cells Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous E6/E7 SV40 LT + hTERT SV40 SV40 LT antigen + hTERT FoxM1B H-RASG12V + p53 knockdown E1A + H-RASG12V + MDM2 Culture propagation/spontaneous MCLR (cyclic hepatotoxin peptide) COX1 (cyclooxygenase1) Culture propagation/spontaneous MET wt or MET mutated FAP (fibroblast activation protein) PTTG1 RPMS1 HER2 ( exon 16) CnB (calcineurin B subunit) VEGF111, 121, and 165 CCK2R mutated ESM1 CD74 HCCR1 hBD3 ( -defensin 3) PDX1 ROBO1 SV40 LT and st, p110 , RASG12V WNT1 c-MYC transcription factor MYCT58A EphA2 ESE1 transcription factor B-crystallin CD8-IGF-IR chimera hGH (human growth hormone) EGFR + c-SRC HOXA1 transcription factor HER2V664E ErbB2 H-RAS mutated or ErbB2 FGF7 (fibroblast growth factor) PDGF (platelet derived growth factor) [199] [200] [10] [24] [201] [202] [203] [25] [204] [205] [206] [207] [208] [209] [210] [211] [212] [213] [214] [215] [216] [217] [26] [48] [27] [218] [219] [220] [221] [222] [109] [223] [137] [224] [225] [226] [227] 51ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells Balb/c 3T3 mouse fibroblasts) cell line in mono- layer culture is common event especially if cells were allowed to reach high density in routine passages (transformation of a diploid line of rat liver cells is also accelerated by the constraint of confluence). It also occurs in low density pas- sages supplemented with low concentrations of serum. If however the cells are kept continuously and rapidly multiplying at low density in high se- rum concentration, not only do they remain non- transformed but they gradually lose the capacity for transformation under standard conditions [310–315]. Grown at different growth conditions (confluence, concentrations of serum) karyotypes of NIH3T3 cells analyzed at different passages (24, 253 and 385 passages) showed that although the chromosome complement of each of the pas- sages was near triploid/hypotetraploid (76 ± 2.65, 74 ± 3.2 and 72 ± 2.3, respectively, instead of the normal 40 chromosomes in mice), there were more marker (i.e., abnormal) chromosomes in passage 385 cells than in the earlier passages [312]. Moreover, every one of the karyotyped cells of each passage was unique in the precise distribution of chromosomes. These results sug- gest that passaging and culture conditions can in- fluence on aneuploid karyotype of NIH3T3 cells. It worth recalling that 293 cells also retained near triploid karyotype through more 100 doublings (6 months in culture) but modal number of chro- mosomes ranged from 62 to 70, that is, there are cell populations inside cell line that differ from each other [304]. In fact, genotypic and pheno- typic variants constantly appear in the cell line populations. In addition to the passage number and the media, the selection of variants is also modulated by the temperature, humidity, and CO2 concentration. Some cells can occur to be Table 5 NIH3T3 cells transformation Indications. mAChR – muscarinic acetylcholine receptor; CDC42HsF28L – full GTPase activity but sponta- neous GTP-GDP exchange; EEF1A2 – protein elongation factor 1A2; EGFR – epidermal growth factor receptor; F-LANa – a member of Derlin family; G o Q205L and G q Q209L – lack of guanosine triphosphatase (GTPase) activity; G6PD – glucose-6-phosphate dehydrogenase; HCCR1 – human cervical cancer oncogene 1; HCCRBP1 – human cervical cancer oncogene binding protein 1; IMUP1 and 2 – immortalization-up-regulated protein 1 and 2; LIN28 and LIN28B – the RNA-binding proteins that block let-7 precursors from being processed to mature miRNAs and consequently derepress let-7 target genes; Matrigel – extract of basement-membrane proteins; Midkine – a heparin-binding growth factor; Mina53 – Myc-induced nuclear antigen; PAR - prostate androgen regulated; PDGF – platelet-derived growth factor; RET – receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor family; STAT3-C – constitutively dimerizable STAT3. Transforming agent Ref. Transforming agent Ref. mAChR AKT1myr AzI (antizyme inhibitor) BCR-ABL + IL3R BI1 (Bax inhibitor 1) CDC42Hs (F28L) Cyclin T1 EEF1A2 EGFR mutant FGF (fibroblast growth factor) F-LANa G q Q209L, wtG q, G o Q205L G6PD HCCR1 HCCRBP1 HPV E7 truncated IMUP1 and 2 [228] [229] [230] [231] [232] [233] [234] [235] [236] [237–239] [240] [241–243] [244] [213] [245] [246] [247] KIT (stem cell factor receptor) Lin28, Lin28B Matrigel Midkine Mina53 MUC4 (mucin) Nanog transcription factor Ornithine decarboxylase PAR PDGF Pleiotrophin Polyamines RET mutants c-SRC + nuclear oncogenes v-SRC v-SRC, STAT3-C 14-3-3 [248] [249] [250] [251] [252] [253] [254] [255, 256] [257] [258, 259] [260] [261] [262] [263] [264] [265] [266] 52 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan more physiologically advantaged to grow during culture monolayer constrains (e.g. confluence). Subsequent selection and clonal propagation of such cells can progressively replace the rest more growth restrained cells leading eventually to full replacement with the cell population with superior growth properties [316]. Selection explains why culturing 52 passages retained 293 cells growth restrained (cells did not form tumors in mice), whereas additional only 13 passages were enough to make cells fully transformed. To the point, au- thors stated that 293 cells were propagated as the cells grown to a 90 % monolayer [309]. Trans- formation can arise by the continuous fluctuation of growth states within cells, accompanied by the progressive selection of those states best suited to function under the selecting constraint [310–315]. The selection may foster cells carrying alterations that confer the capability to proliferate and survive more effectively than their neighbors [198]. Cell- cell contact interactions can conditionally deter- mine suppression or selection of the neoplastic phenotype [315]. It is selection that plays a major role in the spontaneous neoplastic transformation of cells in culture [310–316]. Selection and evolution of cells in vitro and in vivo is universe phenomenon. Nielsen and Briand [317] demonstrated chromosome abnormalities and karyotypic evolution in a nontumorigenic (tested in nude mice) and noninvasive (tested in vitro), spontaneously immortalized cell line HMT-3522, derived from a fibrocystic breast le- sion. During 205 passages, gain and loss of mark- ers, loss of normal chromosomes, and duplica- tion of the chromosome complement could be demonstrated. The variability increased during in vitro growth. This variability led to cells with different growth capacities from which sidelines might be selected and become stem lines. Selec- tion in both directions (non-tumorigenic cells to tumorigenic and vice versa) was also described [154]. This work is of special interest and all ob- servations documented by authors are presented here. The karyotypic changes were associated with the spontaneous acquisition of tumorigenic- ity in an immortalized human bronchial epithe- lial cell line NL20, which had been established by transfection of human bronchial epithelial cells with the SV40 T-antigen. When cells from passage 184 were inoculated into nude mice, a transplantable tumor was obtained. Subsequent passages of the NL20 cells in vitro did not yield further tumors by passage 205. Furthermore, the original tumorigenic NL20T cells lost the neo- plastic phenotype after 25 passages in vitro and reverted to the nontumorigenic karyotype ob- served at passage 189. In contrast to the loss of the tumorigenic phenotype and karyotype, which occurred with in vitro passaging of the original tumor, when the NL20T cells were pas- saged in other nude mice, they continued to give rise to tumors; cells from the secondary tumors (NL20T-A cells) maintained a stable karyotype and remained tumorigenic even after 64 passages in vitro. A mixture of 10 % tumorigenic NL20T- A and 90 % nontumorigenic NL20 cells formed tumors in nude mice when cultured in vitro on fibronectin, but not on plastic; cytogenetic analy- sis demonstrated that the tumors and cell cultures were composed of tumorigenic NL20T-A cells, whereas cells cultured on plastic were identical to the nontumorigenic NL20 cells. Thus, neoplastic transformation in original cell line arose from in vivo selection of a small mutant clone, which had arisen in culture and was subsequently selected in vivo but was lost in in vitro culture [154]. The degree of karyotype heterogeneity determines selection rate and correlates with tumor latent period [318]. The karyotypes of tumors formed by spontaneously transformed Chinese hamster cells of high tumorigenic potential after a short latent period were similar to each other and to the injected cells. The karyotypes of tumors from cells of low tumorigenic potential and long latent periods were diverse, however. No chromosome aberration was common to every tumor. These results suggested that preneoplastic cells whose phenotypes were not directly capable of tumor formation could progress in vivo and that karyo- type instability played an important role in pro- viding cell variants for tumor progression [318]. NIH3T3 cells were fully transformed (showed both anchorage independent growth in soft agar and tumor formation in mice) by a number of transforming agents depicted in Table 5. Actu- ally, as Rubin emphasized «the effectiveness of the NIH3T3 cell line as a target for demonstrat- ing the transforming capacity of oncogenes de- pends on its partially transformed state, which needs only a nudge from an added oncogene to 53ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells progress to more advanced transformation» [315, 319]. Unfortunately, only several works traced karyotype changes accompanied by oncogene transformation. For example, karyotypic analysis of parent NIH3T3 cells and NIH3T3 contain- ing an activated N-RAS oncogene showed that, although the modal chromosome number was comparable for both cell types, number of un- stable chromosomes and forms of abnormalities were different [320]. Another work demonstrated that parental NIH3T3 cells contained 71 chro- mosomes (hypotetraploid), whereas EJ-NIH3T3 (NIH3T3 cell line carrying the transfected human activated H-RAS sequence of EJ human blad- der carcinoma cells) contained 60 chromosomes (triploid) [321]. Moreover, when the latter cells were treated with mutagenes (ethyl methane- sulfonate and 8-azaguanine) and mutant clones were selected, they were resistant to retransfor- mation by Kirsten sarcoma virus, DNA from EJ- NIH 3T3 cells, H-RAS, v-SRC, v-MOS, simian virus 40 large T-antigen, or polyomavirus middle T antigen [321]. Karyotype analysis showed that resistant clones had hyperpentaploid karyotypes (103 ± 9.7 chromosomes) [321]. Transfection of vector containing the mitochondrial D-loop gene from colorectal cancer cell line SW480 into NIH3T3 cells resulted in that NIH3T3 cells had significantly greater percentage of multi- ploid and aberrant chromosomes than control NIH3T3, and this correlated with ability to form colonies in soft agar [322]. Also, other group of investigations suggests that for stable transforma- tion profound changes in genome must occur [232, 238, 246, 323]. MCF10A cell line is spontaneously immortal- ized human mammary epithelial cells with near- diploid karyotype harboring a number of chro- mosome abnormalities. Besides being frequently used in in vitro transformation assays (Table 4), MCF10A cell line was used for comprehensive analysis of the MCF10A series of cell lines rep- resenting progression towards obvious malignancy [165, 324]. The MCF10A progression model con- sists of three directly derived cell lines: the spon- taneously immortalized MCF10A cells (do not show any characteristics of invasiveness or tumor formation), MCF10AT1 cells (MCF10A cells transformed by H-RAS), and MCF10CA1a cells (obtained from tumor in immunodeficient mice after xenograft transplantation of MCF10AT1 cells) [165, 324]. 47 chromosomes were found in MCF10A (gained additional chromosome 8) and the MCF10AT1 cell lines (additional chro- mosome 8 was deleted, but chromosome 9 was gained), whereas the malignant MCF10CA1a cell line had 50 chromosomes [165]. Four marker chromosomes were identified in MCF10A and MCF10AT1 and nine in the malignant MCF- 10CA1a cell line [165]. Spectral karyotyping anal- ysis showed that the premalignant MCF10AT1 gained additional translocations to the MCF10A, whereas the malignant MCF10CA1a had more translocations extra to both MCF10A and MC- F10AT1 [165]. Array comparative genome hy- bridization (aCGH) showed that MCF10A had a number of gains and losses of different chro- mosome regions and progression towards full ma- lignancy was accompanied by much more severe genomic aberrations. Importantly, regions of ge- nomic loss/gain overlapped only partially among these three cell lines [165]. Another investigation exploiting the same model (MCF10A series of cell lines) confirmed the stepwise genome changes ac- companying progression to full malignancy [324]. Moreover, combining SNP array with Gene Array authors showed correlation between DNA copy number gains and increased expression levels for genes located in these regions [324]. Analysis of tumorigenic potential of established seven VERO cell line strains (African green mon- key kidney cells, the normal chromosome number is 60), of which 1 strain was hypotetraploid and the rest strains were hypodiploid, and 3 strains of HeLa cell line (all strains were hyperdiploid) showed that the cell strains were comparatively stable in terms of their heritable characters [325]. There were only little significant changes between passages but the tumorigenicity of strains was different among dif- ferent karyotypic cells (from 7 VERO strains 2 ap- peared tumorigenic: 73 ± 3, and 68 ± 3 or 65 ± 4, hyperdiploids and 5 were nontumorigenic: 54 ± 2, 55 ± 2, 54 ± 2, 54 ± 2, 54 ± 2, all hypodiploids), all HeLa strains were hyperdiploid and tumori- genic [325]. The chromosome number variation of strains had positive relationships with their car- cinogenesis and the chromosome number varia- tion of cell line could be significantly changed when it developed to tumor in nude mice [325]. These observations were confirmed in experiments 54 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan with other cell cultures. Meningioma cells with multiple chromosomal abnormalities grew rapidly in vitro and induced tumors in 49 of 50 animals, whereas with simple karyotypes (less or 1 chromo- somal abnormality) grew slowly in vitro and gave small, nongrowing tumors in mice [326]. Also, the average number of chromosomes in 293N cells (a subline of just obtained 293 cells in 1977 year de- rived from a tumor developed in a nude mouse) was significantly lower than for the parental line [78]. Thus, progression of immortalized or pre-neo- plastic cells towards obvious malignancy is always accompanied by karyotype changes. The degree and diversity of karyotype changes determine tumori- genic potential of cell culture and latency period of tumor formation necessary for creation and/or selection of the most competitive malignant cells. Gene copy number, mRNA and protein lev- els relationships in tumor cells. Actually, primary tumor cells and cancer cell lines are always polip- loid/aneuploid, and have karyotypes ranging from 40 to 60 but occasionally exceeding 70 or more chromosomes [327]. Moreover, numerical large- scale and focal chromosome aberrations (losses/ gains/deletions/ translocations) were found in all samples of each type and subtype of tumors ana- lyzed up to now (Table 6). Roschke et al. [328] using spectral karyotyping provided a description of the chromosomal complement of the NCI-60 cell line panel developed by the National Cancer Institute (NCI) for in vitro anticancer drug screen- ing and reflecting diverse cell lineages (lung, renal, colorectal, ovarian, breast, prostate, central nervous system, melanoma, and hematological malignan- cies). 23 cell lines were identified as near-diploid (a chromosome modal number between 35 and 57), 22 as near-triploid (the chromosome modal num- ber between 58 and 80), 13 as near-tetraploid (a chromosome modal number between 81 and 103), and 1 as near-pentaploid (chromosome modal number between 104 and 126) on the basis of the International System for Chromosome Nomen- clature. The range of numerical changes (clonal chromosome gains and losses) ranged from 1 to 28. Number of structurally rearranged chromosomes (contained translocations, deletions, duplications, insertions, inversions, or homogeneously staining regions) ranged from 1 to 45 (38 cell lines had 10 and more structurally rearranged chromosomes). In addition, in 24 of the 59 cell lines ploidy heteroge- neity was found (i.e., if the majority of cells had a near-triploid karyotype there might be an additional small populations of cells with a near-pentaploid or near-hexaploid count, or, in few cases, with a near-diploid count). Chromosome numerical and structural heterogeneity between cells in the same cell line was also documented [328]. Later, this NCI-60 cell line panel was used to elucidate correlation between gene copy num- ber and mRNA levels for the same gene. The data showed a generally positive correlation be- tween a given gene’s copy number and its ex- pression at the mRNA level supporting the gen- eralization that DNA copy number is one factor (among others) that can influence gene expres- sion [329]. In another work authors performed a global analysis of both mRNA and protein levels based on sequence-based transcriptome analysis (RNA-seq) and SILAC-based mass spectrom- etry analysis [330]. The study was performed in three functionally different human cell lines (the glioblastoma cell line U251MG, the epi- dermoid carcinoma cell line A431 and the osteo- sarcoma cell line U2OS). The changes of mRNA and protein levels in the cell lines using SILAC and RNA ratios showed high correlations, even though the genome-wide dynamic range was sub- stantially higher for the proteins as compared with the transcripts [330]. Also, there was a moderate but significant correlation between global mRNA (RNA-seq) and protein levels (SILAC) of 1710 genes affected by amplification or deletion (SNP and CGH arrays) in seven human metastatic mela- noma cell lines [331]. Whether does it mean that the end point of gene expression, the level of pro- tein, is affected proportionally to copy number of gene in tumor cells? Measurement of expression levels of 6735 proteins was directly compared to the gene copy number in MCF7 breast cancer cell line [332]. Authors found that in the majority of cases, there was no direct correspondence between the gene copy number change and the correspond- ing protein change. Nevertheless, proteins encoded by amplified oncogenes were often overexpressed, while adjacent amplified genes, which presumably did not promote growth and survival, were attenua- ted [332]. Furthermore, authors revealed that the proteins of such complexes as the proteasome, ribo- some, spliceosome, and NADH dehydrogenase al- ways maintained equal protein ratios, despite varia- 55ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells tion in the gene copy number of their subunits. This was strictly true for the core complexes components, but to a lesser degree for peripheral proteins, which could also be involved in other processes [332]. In- terestingly, levels of protein expression in aneuploid yeast strains largely scale with chromosome copy numbers, following the same trend as that observed for the transcriptome [333]. Thus, eventually to be definitely concluded, relationships between gene copy number, mRNA level and protein level of indi- vidual genes across the whole cancer genome should be analyzed. It would give the comprehensive under- standing to which degree regulation of gene expres- sion on different levels operates in tumor cells and which groups of genes are predominantly imposed on such regulation. Inter- and intratumor heterogeneity. It is sup- posed that common (clonal) chromosome changes are the «drivers» of neoplastic transformation whereas rare chromosome changes (non-clonal) are likely the «passengers» in this process, which may be either nonfunctional or functional but con- stitute secondary events [3, 374, 375]. Nevertheless, non-clonal aberrations reflect the significant tumor feature: genome/chromosome instability and, as a consequence, inter- and intratumor genome het- erogeneity [376]. Importantly, the main determi- nant of the ability of a population to evolve is the extent of heritable variation within the population [377]. Numerous studies have proved that intra-tu- mor genetic heterogeneity/clonal diversity is a key force driving transformation and tumor evolution (Stepanenko and Kavsan, in preparation). Nobusawa et al. [374] have analysed by aCGH separate tumor areas of 14 primary glio- blastomas (total, 41 tumor areas). They revealed Table 6 Tumors and cell lines with multiple chromosomal abnormalities Indications. aCGH – array comparative genome hybridization; SNP array – single nucleotide polymorphism array; Sequencing – massively parallel paired-end sequencing; MLDPA – multiple ligation-dependent probe amplification. Cancer type Method Number of samples Ref. Acute myeloid leukemia Bladder cancer Bladder carcinomas Breast cancer Cervical carcinomas Colorectal carcinomas and adenomas Endometrial carcinomas and carcinosarcomas Ewing’s cancer Gastric cancer Germ cell cancer Glioma Head and neck squamous cell carcinomas Lung cancer Myelodysplastic syndromes and related myeloid malignancies Oral carcinomas Oropharynx and hypopharynx squamous cell carcinomas Ovarian epithelial tumors Pancreatic carcinomas Prostate tumours Thyroid carcinomas and adenomas 29 different tissues 26 different tissues aCGH aCGH aCGH aCGH, SNP array, sequencing, aCGH, SNP array aCGH, SNP array aCGH and karyotyping aCGH, spectral karyotyping aCGH aCGH aCGH, SNP array, karyotyping aCGH Sequencing, SNP array, karyotyping SNP array, karyotyping aCGH aCGH aCGH aCGH, sequencing Sequencing aCGH aCGH SNP array 17 22 109 1143 40 129 82 7 31 24 248 43 80 430 60 20 47 37 7 28 598 3131 [334 ] [335] [336, 337] [338–342] [343, 344] [345–348] [349] [350] [351] [352] [353–359] [360, 361] [362, 363] [364] [365] [366] [367] [368, 369] [370] [371] [372] [373] 56 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan that chromosomal imbalances significantly dif- fered among glioblastomas. In addition, there were numerous tumor area-specific genomic im- balances. Analysis of disseminated single cells in minimal residual disease has shown that there is a high level of genomic heterogeneity within indi- vidual lesions as well as between primary tumors and metastatic cells [376]. Giving comments on reports of breast cancer genomes analyses with high-throughput genomics thechnics [339, 342], Swanton et al. [378] concluded that results from these studies had revealed «perplexing breast can- cer genome complexity with very few aberrations occurring in common between breast cancers. In addition, such complexity is compounded by evi- dence of profound genomic heterogeneity within individual breast tumors (intratumoral hetero- geneity), where multiple tumor subpopulations have been identified, each with distinct genomic profiles heterogeneity occurring within individual breast cancers». Moreover, recurrent tumors al- ways show appearance of new chromosome im- balances and gene mutations distinct from those, which were observed in most cells of a primary tumor but could be harbored by a small group of cells within a primary tumor or acquired de novo [371, 377, 380–389]. Intratumor genomic heterogeneity is created and fostered by chromosome instability (CIN. Al- though defects in chromosome cohesion, kinet- ochore-microtubule misattachments, assembly of multipolar mitotic spindles [182, 390–396], translocations containing breakpoints within fra- gile sites [397], satellite repeats in heterochro- matin [398], cell-in-cell formation by entosis (as a result, cytokinesis frequently fails, generating binucleate cells that produce aneuploid cell lin- eages) [399], random fragmentation of the entire chromosome (chromothripsis) in which chromo- somes are broken into many pieces and then randomly stitched back together [400, 401] can contribute to CIN, in cancer cells mechanism of centrosome amplification and clustering is proposed to be the major contributor to CIN. Importantly, there is compelling evidence that diverse oncogenes or carcinogenes induce cen- trosome deregulation and CIN (Stepanenko and Kavsan, in preparation). Inter- and intratumor heterogeneity of gene mutations was also revealed by sequencing of all protein coding genes in several solid tumors, in- cluding glioblastomas, colorectal, pancreatic and breast cancers. It was found that individual solid organ tumors harbor approximately 40–80 clonal mutations per tumor in the coding regions of dif- ferent genes, and although a few of these genes are mutated in a high proportion of tumors, the preva- lence at which the majority are mutated among dif- ferent tumors of the same cancer type is low [376, 377, 402]. 2576 somatic mutations were identified across 1800 megabases of DNA representing 1507 coding genes from 441 tumors comprising breast, lung, ovarian and prostate cancer types and sub- types [403]. Authors found that mutation rates and the sets of mutated genes varied substantially across tumor types and subtypes. Results of sequencing COLO-829 cancer cell line derived, before treat- ment, from a metastasis of a malignant melanoma demonstrated a total of 292 somatic base substitu- tions in protein-coding sequences [404]. As Fox et al. stated «each tumor displays a unique and diverse profile of mutated genes, but no new prevalently mutated genes are identified... Within an individual neoplasm, a few mutations are present throughout the population, a greater number are present in mi- nority subclones, and the majority is found in only one or a few cells» [376]. Actually, if to calculate all abnormalities of noncoding genome regions, there are usually between 1000 and 10000 somatic sub- stitutions in the genomes of most adult tumors, in- cluding breast, ovary, colorectal, pancreas cancers, and glioma [3]. All these data imply that tumors are really «oncogene addicted», but it has not revealed a possible «Achilles’ heel» within the cancer cell that can be exploited therapeutically [3, 405], be- cause «instead of long-anticipated common muta- tions, a large number of stochastic gene mutations were detected for each individual with the same cancer type» [406]. Chromosome instability and drug resistance. Cancer cells rapidly acquire resistance against numerous cytotoxic drugs or are even intrinsically resistant [379]. The chromosomes of cancer cells are extremely unstable compared to those of normal cells: 1 in 100 highly aneuploid human cancer cells loses or gains or rearranges a chromosome per cell generation [407]. Genetic variation within a cancer cell population reflects dynamics of clonal evolution and, importantly, serves as a reservoir of genetic diversity from which therapy-resistant 57ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells clones may arise [376]. In vitro studies have con- firmed that CIN cells acquire multidrug resistance at an accelerated rate compared with diploid cells resulting from the selection pressure influenced by drug exposure. Moreover, mouse CIN cells be- came multidrug resistant even after deletion of all known multidrug resistance genes [319, 379]. To identify distinct therapeutic approaches to specifi- cally limit the growth of CIN tumors, Lee et al. [408] focused on a panel of colorectal cancer cell lines, previously classified as either chromosom- ally unstable, CIN (+), or diploid/near-diploid, CIN (–), and treated them individually with a library of kinase inhibitors targeting components of signal transduction, cell cycle, and transmem- brane receptor signaling pathways. CIN (+) cell lines displayed significant intrinsic multidrug re- sistance compared with CIN (–) cancer cell lines, and this seemed to be independent of somatic mu- tation status and proliferation rate [408]. According to Duesberg et al., karyotype plays the central role in drug resistance [379]. «When cancer cells acquire resistance against drugs, they acquire new karyotypic alterations and/or they lose old ones». Indeed, gene expression profiles of drug resistant cells differ from those of parental drug sensitive cells in the over- or underexpression of hundreds to thousands genes [379]. Comparison of the structures of the puromycin resistance- specific chromosomal alterations in four different human colon cancer lines indicates, that most but not all of resistance-specific chromosomal alterations were unique for each cancer cell [379]. Drug resistance correlates with chromosomal alterations [319, 407]. It is generated de novo in cancer cells by chromosome re-assortments [379]. The resulting level of resistance is proportional to the numbers of resistance-specific chromosomal alterations or «tumor heterogeneity» [379]. In the presence of cytotoxic drugs resistance-specific alterations are selected from the resultining vari- ants by classical Darwinian mechanisms [379]. Ñhromosome instability, intertumoral and intra- tumoral heterogeneity present a challenge to per- sonalized therapeutic approaches [378]. Conclusion. The intense searching for the ab- normal genes influencing the development of hu- man cancer revealed about 200000 somatic mu- tations in cancer genomes (COSMIC database) since the first somatic mutation that was found in H-RAS the quarter of a century ago [198]. Hun- dreds of genes are being considered and dozens genes/proteins have been used already as poten- tial drug targets in clinical trials. However, at present benefits from oncogene directed therapy are still moderate. Large-scale tumor genome se- quencing have failed to reveal «universal» can- cer genes and, instead, «large numbers of diverse mutations have been identified dominating the cancer genome landscape» [406]. «A future of multiple targeted therapies and patient stratifica- tion, based on a mutational signature of defined key genes for each cancer type, seems less hope- ful than initially anticipated» [376]. Now it is clear that «cancer progression is a stochastic process both at the genome and gene level, and is not a stepwise process defined by sequential genetic aberrations. Stochastic pro- cess frequently occurs prior to the key stages of immortalization, transformation and metasta- sis and results in inability to detect type- and stage-specific recurrent aberrations in solid tu- mors» [406]. Numerous somatic rearrangements, including whole chromosome and copy number gains and losses, chromosome translocations, and gene mutations participate in establishing the malignant cell phenotype. «Multiple rounds of proliferation, often counter-balanced by cell death, are required to produce macroscopic tu- mors, and genomic instability, observed in most cancers, is expected to constantly produce new mutations, which serve as raw material on which tumor evolution can work» [409]. The selection may weed out cells that have acquired deleteri- ous mutations or it may foster cells carrying al- terations that confer the capability to proliferate and survive more effectively than their neighbors [198]. A single cell can acquire a set of suffi- ciently advantageous mutations that allows it to proliferate autonomously, invade tissues and me- tastasize [198]. CIN is the most common genetic abnormality of cancer cells and tumorigenic cell lines. The fre- quent losses and gains of whole chromosomes dur- ing cell divisions in CIN cancer cells trigger rapid alterations in gene dosage [379]. In vitro studies have confirmed that CIN cells acquire multi-drug resistance at an accelerated rate compared with diploid cells [379]. Furthermore, although most cancers are of monoclonal origin, the expansion of 58 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 A.A. Stepanenko, V.M. Kavsan the population size, which occurs after malignant transformation, coupled with the constant acquisi- tion of mutations promotes the diversion into sub- clones and a dramatic increase in genetic tumor heterogeneity [377]. High genetic heterogeneity of tumors means high probability of pre-existent clones that are resistant to therapeutic intervention and can be selected by therapy resulting in therapy failure [409]. Severe genome rearrangements and intratumoral heterogeneity challenge oncogene di- rected therapy, while chromosome instability and karyotype evolution make each tumor «a moving rather than frozen» target. Thus, a main driver of evolutionary adaptation during drug treatment is the genetic heterogeneity, which is fostered by CIN. New tools are necessary to study heterogeneity and to analyze changes in heterogeneity and clonal composition during drug treatment [410–412]. This would allow new in- sights into these processes and provide the basis to improve therapeutic outcomes based on tumor evolution and the specific targeting of distinct ge- nomic instability mechanisms [377, 413]. This work was supported in part by grant SFFR F46/457-2011 «State key laboratory of molecular and cellular biology» and by budget topic of the Institute of Molecular Biology and Genetics, NAS of Ukraine, «Functional characterization of genes associated with initiation and progression of glial and connective tissue tumors of human brain». À.À. Ñòåïàíåíêî, Â.Ì. Êàâñàí ÈÌÌÎÐÒÀËÈÇÀÖÈß È ÇËÎÊÀ×ÅÑÒÂÅÍÍÀß ÒÐÀÍÑÔÎÐÌÀÖÈß ÝÓÊÀÐÈÎÒÈ×ÅÑÊÈÕ ÊËÅÒÎÊ ×òîáû ñòàòü ïîëíîñòüþ òðàíñôîðìèðîâàííîé îïó- õîëåâîé êëåòêîé, íîðìàëüíàÿ êëåòêà äîëæíà ïðå- îäîëåòü ðÿä âíóòðåííèõ êëåòî÷íûõ áàðüåðîâ è ïðè- îáðåñòè áîëüøîå ÷èñëî õðîìîñîìíûõ èçìåíåíèé. Ïåðâûì è íåîáõîäèìûì øàãîì â çëîêà÷åñòâåííîé òðàíñôîðìàöèè ÿâëÿåòñÿ ïðåîäîëåíèå ñòàðåíèÿ, èëè èììîðòàëèçàöèÿ êëåòêè. Èììîðòàëèçèðîâàííûå êëåòêè ìîãóò áåñêîíå÷íî äîëãî ïðîëèôåðèðîâàòü â ïðèñóòñòâèè ðîñòîâûõ ôàêòîðîâ è ïèòàòåëüíûõ âå- ùåñòâ. Èììîðòàëèçèðîâàííûå êëåòêè íèêîãäà íå èìåþò íîðìàëüíîãî äèïëîèäíîãî êàðèîòèïà, xoòÿ âî âðåìÿ ðîñòà ïîäâåðãàþòñÿ êîíòàêòíîìó èíãèáè- ðîâàíèþ, íå ôîðìèðóþò êîëîíèé â ìÿãêîì àãàðå (ò.å. çàâèñèìûé îò ïîäëîæêè ðîñò) è íå ôîðìèðóþò îïóõîëåé ïðè ââåäåíèè èììóíîäåôèöèòíûì ìûøàì. Âñå ýòè ñâîéñòâà ìîãóò áûòü ïðèîáðåòåíû ñ äîïîëíèòåëüíû- ìè õðîìîñîìíûìè èçìåíåíèÿìè. Ìíîæåñòâåííûå ãå- íåòè÷åñêèå èçìåíåíèÿ, âêëþ÷àÿ ïðèîáðåòåíèå/ïîòå- ðþ öåëûõ õðîìîñîì èëè îòäåëüíûõ ó÷àñòêîâ/ëîêó- ñîâ, òðàíñëîêàöèþ õðîìîñîì è ãåííûå ìóòàöèè, íåîáõîäèìû äëÿ óñòàíîâëåíèÿ òðàíñôîðìèðîâàííî- ãî ôåíîòèïà. Ïðîöåññ êëåòî÷íîé òðàíñôîðìàöèè äî- ñòàòî÷íî õîðîøî èçó÷åí íàêëåòî÷íûõ êóëüòóðàõ in vitro. Áîëüøèíñòâî ýêñïåðèìåíòîâ, âûÿâèâøèõ òðàíñôîðìèðóþùóþ ñïîñîáíîñòü ãåíîâ (îíêîãåíîâ), íàäýêñïðåññèðîàííûõ è/èëè ìóòèðîâàííûõ â îïóõî- ëÿõ, áûëî âûïîëíåíî ñ èñïîëüçîâàíèåì òàêèõ êëåòî÷- íûõ êóëüòóð, êàê ìûøèíûå ýìáðèîíàëüíûå ôèáðîáëà- ñòû (MEFs), ìûøèíàÿ êëåòî÷íàÿ ëèíèÿ ôèáðîáëàñòîâ NIH3T3, êëåòî÷íàÿ ëèíèÿ ÷åëîâå÷åñêîé ýìáðèî- íàëüíîé ïî÷êè 293 (293 êëåòêè) è ýïèòåëèàëüíûå êëåòî÷íûå ëèíèè ìîëî÷íîé æåëåçû ÷åëîâåêà (ãëàâ- íûì îáðàçîì, HMECs è MCF10A), êîòîðûå ïðåäñòàâ- ëÿþò ñîáîé èììîðòàëèçèðîâàííûå êëåòêè (êðî- ìå ïåðâè÷íûõ ìûøèíûõ ôèáðîáëàñòîâ) ñ èçìåíåí- íûìè ãåíîìàìè (ïîëè-/àíåóïëîèäû ñî çíà÷èòåëü- íûìè õðîìîñîìíûìè ïåðåñòðîéêàìè) è ñêëîííûå ê ïîëíîé çëîêà÷åñòâåííîé òðàíñôîðìàöèè ïðè êóëü- òèâèðîâàíèÿ. Íåäàâíî îáíîâëåííûé ñïèñîê îíêî- ãåíîâ âêëþ÷àåò áîëåå 467 ãåíîâ, êîòîðûå, êàê ïî- ëàãàþò, âîâëå÷åíû â ðàçâèòèå îïóõîëè, êîãäà ñî- îòâåòñòâåííûì îáðàçîì èçìåíåíû (òî÷êîâûå ìóòà- öèè, äåëåöèè, òðàíñëîêàöèè èëè àìïëèôèêàöèè). Îäíàêî èññëåäîâàíèÿ íà ìûøàõ ñâèäåòåëüñòâóþò, ÷òî áîëåå 3000 ãåíîâ ìîãóò âíîñèòü âêëàä â ðàçâèòèå îïóõîëè. Öåëüþ íàñòîÿùåãî îáçîðà ÿâ- ëÿåòñÿ ïîíÿòü ìåõàíèçìû êëåòî÷íîé èììîðòàëè- çàöèè ðàçëè÷íûìè «èììîðòàëèçóþùèìè àãåíòàìè», îíêîãåí-èíäóöèðóåìîé êëåòî÷íîé òðàíñôîðìàöèè èììîðòàëèçèðîâàííûõ êëåòîê è óìåðåííûé îòâåò íà òåðàïèþ èç-çà «ñêëîííîñòè» îïóõîëè ê ïðèîáðåòå- íèþ ìíîãî÷èñëåííûõ ãåííûõ è õðîìîñîìíûõ èçìå- íåíèé, âíóòðè- è ìåæîïóõîëåâîé ãåòåðîãåííîñòè. Î.À. Ñòåïàíåíêî, Â.Ì. Êàâñàí ²ÌÎÐÒÀË²ÇÀÖ²ß ÒÀ ÇËÎßÊ²ÑÍÀ ÒÐÀÍÑÔÎÐÌÀÖ²ß ÅÓÊÀÐ²ÎÒÈ×ÍÈÕ ÊË²ÒÈÍ Ùîá ñòàòè ïîâí³ñòþ òðàíñôîðìîâàíîþ ïóõ- ëèííîþ êë³òèíîþ, íîðìàëüíà êë³òèíà ïîâèííà ïîäîëàòè íèçêó âíóòð³øí³õ êë³òèííèõ áàð’ºð³â ³ ïðèäáàòè âåëèêó ê³ëüê³ñòü õðîìîñîìíèõ çì³í. Ïåð- øèì íåîáõ³äíèì êðîêîì ó çëîÿê³ñí³é òðàíñôîðìàö³¿ º ïîäîëàííÿ ñòàð³ííÿ, àáî ³ìîðòàë³çàö³ÿ êë³òèíè. ²ìîðòàë³çîâàí³ êë³òèíè ìîæóòü íåñê³í÷åííî äîâãî ïðîë³ôåðóâàòè â ïðèñóòíîñò³ ðîñòîâèõ ôàêòîð³â ³ ïîæèâíèõ ðå÷îâèí. ²ìîðòàë³çîâàí³ êë³òèíè ìàéæå í³êîëè íå ìàþòü íîðìàëüíîãî äèïëî¿äíîãî êàð³î- òèïó, òèì íå ìåíø âîíè ï³ä ÷àñ ðîñòó ï³ääàþòüñÿ êîíòàêòíîìó ³íã³áóâàííþ, íå ôîðìóþòü êîëîí³é â ì’ÿêîìó àãàð³ (òîáòî çàëåæíå â³ä ï³äêëàäêè çðîñ- 59ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 2 Immortalization and malignant transformation of eukaryotic cells òàííÿ) ³ íå ôîðìóþòü ïóõëèí ïðè ââåäåíí³ ³ìóíî- äåô³öèòíèì ìèøàì. Âñ³ ö³ âëàñòèâîñò³ ñòàá³ëüíî ìîæóòü áóòè ïðèäáàí³ ç äîäàòêîâèìè õðîìîñîì- íèìè çì³íàìè. Ìíîæèíí³ ãåíåòè÷í³ çì³íè, âêëþ÷à- þ÷è íàáóòòÿ àáî âòðàòó ö³ëèõ õðîìîñîì àáî îêðåìèõ ä³ëÿíîê/ëîêóñ³â, òðàíñëîêàö³ÿ õðîìîñîì ³ ãåíí³ ìó- òàö³¿, º íåîáõ³äíèìè äëÿ âñòàíîâëåííÿ òðàíñôîðìî- âàíîãî ôåíîòèïó. Ïðîöåñ êë³òèííî¿ òðàíñôîðìàö³¿ äîñèòü äîáðå âèâ÷åíèé íà êë³òèííèõ êóëüòóðàõ in vitro. Á³ëüø³ñòü åêñïåðèìåíò³â ç âèÿâëåííÿ òðàíñôîðìóþ÷î¿ çäàòíîñò³ ãåí³â (îíêîãåí³â), íàä- åêñïðåñîâàíèõ òà/àáî ìóòîâàíèõ â ïóõëèíàõ, áóëî âèêîíàíî ç âèêîðèñòàííÿì òàêèõ êë³òèííèõ êóëü- òóð, ÿê ìèøà÷³ åìáð³îíàëüí³ ô³áðîáëàñòè (MEFs), êë³òèííà ë³í³ÿ ìèøà÷èx ô³áðîáëàñò³â NIH3T3, êë³òèííà ë³í³ÿ ëþäñüêî¿ åìáð³îíàëüíî¿ íèðêè 293 (293 êë³òèíè) ³ åï³òåë³àëüí³ êë³òèíí³ ë³í³¿ ìî- ëî÷íî¿ çàëîçè ëþäèíè (ãîëîâíèì ÷èíîì, HMECs ³ MCF10A), ÿê³ ïðåäñòàâëÿþòü ñîáîþ ³ìîðòàë³- çîâàí³ êë³òèíè (êð³ì ïåðâèííèõ ìèøà÷èõ ô³áðî- áëàñò³â) ç³ çì³ííèìè êàð³îòèïàìè (ïîë³-/àíåó- ïëî¿äè ç³ çíà÷íèìè õðîìîñîìíèìè ïåðåáóäîâàìè) ³ ñõèëüí³ äî ïîâíî¿ çëîÿê³ñíî¿ òðàíñôîðìàö³¿ ïðè êóëüòèâóâàíí³. Íåùîäàâíî îíîâëåíèé ñïèñîê îíêî- ãåí³â âêëþ÷àº ïîíàä 467 ãåí³â, ùî çàëó÷åí³, ÿê ââàæàþòü, äî ðîçâèòêó ïóõëèíè, êîëè â³äïîâ³äíèì ÷èíîì çì³íåí³ (òî÷êîâ³ ìóòàö³¿, äåëåö³¿, òðàíñëîêàö³¿ àáî àìïë³ô³êàö³¿). Îäíàê äîñë³äæåííÿ íà ìèøàõ ñâ³ä÷àòü ïðîòå, ùî ïîíàä 3000 ãåí³â ìîæóòü ðî- áèòè âíåñîê ó ðîçâèòîê ïóõëèíè. 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Immortalization and malignant transformation of eucaryotic cells

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