From reverse transcription to human brain tumors
Reverse transcriptase from avian myeloblastosis virus (AMV) was the subject of the study, from which the investigations of the Department of biosynthesis of nucleic acids were started. Production of AMV in grams quantities and isolation of AMV reverse transcriptase were established in the laboratory...
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Інститут молекулярної біології і генетики НАН України
2013
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irk-123456789-1525802019-06-13T01:25:42Z From reverse transcription to human brain tumors Dmitrenko, V.V. Avdieiev, S.S. Areshkov, P.O. Balynska, O.V. Bukreieva, T.V. Stepanenko, A.A. Chausovskii, T.I. Kavsan, V.M. Reviews Reverse transcriptase from avian myeloblastosis virus (AMV) was the subject of the study, from which the investigations of the Department of biosynthesis of nucleic acids were started. Production of AMV in grams quantities and isolation of AMV reverse transcriptase were established in the laboratory during the seventies of the past century and this initiated research on the cDNA synthesis, cloning and investigation of the structure and functions of the eukaryotic genes. Structures of salmon insulin and insulin-like growth factor (IGF) family genes and their transcripts were determined during long-term investigations. Results of two modern techniques, microarray-based hybridization and SAGE, were used for the identification of the genes differentially expressed in astrocytic gliomas and human normal brain. Comparison of SAGE results on the genes overexpressed in glioblastoma with the results of microarray analysis revealed a limited number of common genes. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of glioblastoma. The first experiments on the classification of glioblastomas based on the data of the 20 genes expression were conducted by using of artificial neural network analysis. The results of these experiments showed that the expression profiles of these genes in 224 glioblastoma samples and 74 normal brain samples could be according to the Kohonen’s maps. The CHI3L1 and CHI3L2 genes of chitinase-like cartilage protein were revealed among the most overexpressed genes in glioblastoma, which could have prognostic and diagnostic potential. Results of in vitro experiments demonstrated that both proteins, CHI3L1 and CHI3L2, may initiate the phosphorylation of ERK1/ ERK2 and AKT kinases leading to the activation of MAPK/ERK1/2 and PI3K/AKT signaling cascades in human embryonic kidney 293 cells, human glioblastoma U87MG, and U373 cells. The new human cell line 293_CHI3L1, stably producing chitinase-like protein CHI3L1 was developed and these cells were found to have an accelerated growth rate and could undergo anchorage-independent growth in soft agar which is one of the most consistent indicators of oncogenic transformation. The formation of tumors in rats by 293_CHI3L1 cells evidences that CHI3L1 is an oncogene involved in tumorigenesis. In vitro experiments showed that constitutive expression of CHI3L1 gene promotes chromosome instability in 293 cells. Наукові розробки відділу біосинтезу нуклеїнових кислот розпочато з вивчення зворотної транскриптази вірусу пташиного мієлобластозу (AMV). Протягом сімдесятих років минулого століття у відділі налагоджено виробництво AMV (декілька грамів на рік) та виділення зворотної транскриптази AMV, що дозволило розгорнути роботи з синтезу кДНК, клонування та вивчення структури і функції генів евкаріотів. Упродовж багаторічних досліджень було визначено будову генів інсуліну і родини інсуліноподібних факторів росту (IGF) лосося та їхніх транскриптів. Результати застосування двох сучасних методів – гібридизації мікрочіпів і SAGE – використано для ідентифікації генів, які диференційно експресуються в астроцитарних гліомах і нормальному головному мозку людини. Їхнє порівняння виявило обмежену кількість спільних генів, надекспресованих у гліобластомі. Визначені нами 105 диференційно експресованих генів, спільних для обох методів, можуть бути включені до переліку кандидатів для молекулярного типування гліобластом. Проведено перші експерименти з класифікації гліобластом на основі даних по експресії 20 генів із застосуванням штучної нейронної мережі, які показали, що профілі експресії зазначених генів для 224 зразків гліобластом і 74 зразків нормального головного мозку піддаються кластеризації згідно з картами Кохонена. Серед найекспресованіших у гліобластомі генів, які мають прогностичний і діагностичний потенціал, виявлено гени хітиназоподібних білків CHI3L1 і CHI3L2. Результати експериментів in vitro продемонстрували, що обидва білки – CHI3L1 і CHI3L2 – здатні ініціювати фосфорилювання кіназ ERK1/ERK2 і AKT, що спричиняє активацію сигнальних каскадів PI3K/AKT і MAPK/ERK1/2 в клітинах 293 ембріональної нирки людини, а також у клітинах U87MG і U373 гліобластоми людини. Ідентифіковано нову клітинну лінію людини 293_CHI3L1, яка стабільно продукує хітиназоподібний білок CHI3L1. Знайдено, що ці клітини мають прискорений ріст і можуть рости у м’якому агарі незалежно від прикріплення до поверхні, що є одним із найсуттєвіших показників пухлинної трансформації. Формування пухлин клітинами 293_CHI3L1 у щурів свідчить про те, що CHI3L1 є онкогеном, причетним до канцерогенезу. Експерименти in vitro засвідчили, що конститутивна експресія гена CHI3L1 сприяє хромосомній нестабільності у клітинах 293. Модальне число хромосом у клітинах 293_CHI3L1 відрізняється від такого хромосом у контрольних клітинах 293_pcDNA3.1, трансфікованих «порожнім» плазмідним вектором, і батьківських клітинах 293. Научные разработки отдела биосинтеза нуклеиновых кислот начались с изучения обратной транскриптазы вируса птичьего миелобластоза (AMV). В течение семидесятых годов прошлого века в отделе налажено производство AMV (несколько граммов в год) и выделение обратной транскриптазы AMV, что позволило развернуть работы по синтезу кДНК, клонированию и исследование структуры и функции генов эукариотов. На протяжении многолетних исследований было определено строение генов инсулина и семейства инсулиноподобных факторов роста (IGF) лосося и их транскриптов. Результаты применения двух современных методов – гибридизации микрочипов и SAGE – использованы для идентификации генов, дифференциально экспрессируются в астроцитарных глиомах и нормальном головном мозге человека. Их сравнение выявило ограниченное число общих генов, надэкспрессиро- ванных в глиобластоме. Определенные нами 105 дифференциально экспрессированных генов, общих для обоих методов, могут быть включены в список кандидатов для молекулярного типирования глиобластом. Проведены первые эксперименты по классификации глиобластом на основе данных по экспрессии 20 генов с использованием искусственной нейронной сети, показавшие, что профили экспрессии этих генов для 224 образцов глиобластом и 74 образцов нормального головного мозга могут быть кластеризованы в соответствии с картами Кохонена. Среди наиболее надэкспресированных в глиобластоме генов, имеющих прогностический и диагностический потенциал, обнаружены гены хитиназоподобных белков CHI3L1 и CHI3L2. Результаты экспериментов in vitro продемонстрировали, что оба белка – CHI3L1 и CHI3L2 – могут инициировать фосфорилирование киназ ERK1/ ERK2 и AKT, приводящее к активации сигнальных каскадов PI3K/ AKT и MAPK/ERK1/2 в клетках 293 эмбриональной почки человека, а также в клетках U87MG и U373 глиобластомы человека. Получена новая клеточная линия человека 293_CHI3L1 со стабильной продукцией хитиназоподибного белка CHI3L1. Обнаружено также, что эти клетки обладают ускоренным ростом и могут расти в мягком агаре независимо от прикрепления к поверхности. Такие свойства являются одним из наиболее существенных показателей опухолевой трансформации. Формирование опухолей клетками 293_CHI3L1 у крыс свидетельствует о том, что CHI3L1 является онкогеном, участвующим в канцерогенезе. Эксперименты in vitro показали, что конститутивная экспрессия гена CHI3L1 способствует хромосомной нестабильности в клетках 293. Модальное число хромосом в клетках 293_CHI3L1 отличается от такового хромосом в контрольных клетках 293_ pcDNA3.1, трансфецированных «пустым» плазмидным вектором, и родительских клетках 293. 2013 Article From reverse transcription to human brain tumors / V.V. Dmitrenko, S.S. Avdieiev, P.O. Areshkov, O.V. Balynska, T.V. Bukreieva, A.A. Stepanenko, T.I. Chausovskii, V.M. Kavsan // Вiopolymers and Cell. — 2013. — Т. 29, №. 3. — С. 221-233. — Бібліогр.: 99 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00081C http://dspace.nbuv.gov.ua/handle/123456789/152580 577.21:577.214.622 + 616-006.484.04 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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Reviews Reviews |
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Reviews Reviews Dmitrenko, V.V. Avdieiev, S.S. Areshkov, P.O. Balynska, O.V. Bukreieva, T.V. Stepanenko, A.A. Chausovskii, T.I. Kavsan, V.M. From reverse transcription to human brain tumors Вiopolymers and Cell |
| description |
Reverse transcriptase from avian myeloblastosis virus (AMV) was the subject of the study, from which the investigations of the Department of biosynthesis of nucleic acids were started. Production of AMV in grams quantities and isolation of AMV reverse transcriptase were established in the laboratory during the seventies of the past century and this initiated research on the cDNA synthesis, cloning and investigation of the structure and functions of the eukaryotic genes. Structures of salmon insulin and insulin-like growth factor (IGF) family genes and their transcripts were determined during long-term investigations. Results of two modern techniques, microarray-based hybridization and SAGE, were used for the identification of the genes differentially expressed in astrocytic gliomas and human normal brain. Comparison of SAGE results on the genes overexpressed in glioblastoma with the results of microarray analysis revealed a limited number of common genes. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of glioblastoma. The first experiments on the classification of glioblastomas based on the data of the 20 genes expression were conducted by using of artificial neural network analysis. The results of these experiments showed that the expression profiles of these genes in 224 glioblastoma samples and 74 normal brain samples could be according to the Kohonen’s maps. The CHI3L1 and CHI3L2 genes of chitinase-like cartilage protein were revealed among the most overexpressed genes in glioblastoma, which could have prognostic and diagnostic potential. Results of in vitro experiments demonstrated that both proteins, CHI3L1 and CHI3L2, may initiate the phosphorylation of ERK1/ ERK2 and AKT kinases leading to the activation of MAPK/ERK1/2 and PI3K/AKT signaling cascades in human embryonic kidney 293 cells, human glioblastoma U87MG, and U373 cells. The new human cell line 293_CHI3L1, stably producing chitinase-like protein CHI3L1 was developed and these cells were found to have an accelerated growth rate and could undergo anchorage-independent growth in soft agar which is one of the most consistent indicators of oncogenic transformation. The formation of tumors in rats by 293_CHI3L1 cells evidences that CHI3L1 is an oncogene involved in tumorigenesis. In vitro experiments showed that constitutive expression of CHI3L1 gene promotes chromosome instability in 293 cells. |
| format |
Article |
| author |
Dmitrenko, V.V. Avdieiev, S.S. Areshkov, P.O. Balynska, O.V. Bukreieva, T.V. Stepanenko, A.A. Chausovskii, T.I. Kavsan, V.M. |
| author_facet |
Dmitrenko, V.V. Avdieiev, S.S. Areshkov, P.O. Balynska, O.V. Bukreieva, T.V. Stepanenko, A.A. Chausovskii, T.I. Kavsan, V.M. |
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Dmitrenko, V.V. |
| title |
From reverse transcription to human brain tumors |
| title_short |
From reverse transcription to human brain tumors |
| title_full |
From reverse transcription to human brain tumors |
| title_fullStr |
From reverse transcription to human brain tumors |
| title_full_unstemmed |
From reverse transcription to human brain tumors |
| title_sort |
from reverse transcription to human brain tumors |
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Інститут молекулярної біології і генетики НАН України |
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2013 |
| topic_facet |
Reviews |
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http://dspace.nbuv.gov.ua/handle/123456789/152580 |
| citation_txt |
From reverse transcription to human brain tumors / V.V. Dmitrenko, S.S. Avdieiev, P.O. Areshkov, O.V. Balynska, T.V. Bukreieva, A.A. Stepanenko, T.I. Chausovskii, V.M. Kavsan // Вiopolymers and Cell. — 2013. — Т. 29, №. 3. — С. 221-233. — Бібліогр.: 99 назв. — англ. |
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Вiopolymers and Cell |
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UDC 577.21:577.214.622 + 616-006.484.04
From reverse transcription to human brain tumors
V. V. Dmitrenko, S. S. Avdieiev, P. O. Areshkov, O. V. Balynska,
T. V. Bukreieva, A. A. Stepanenko, T. I. Chausovskii, V. M. Kavsan
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnogo Str., Kyiv, Ukraine 03680
dmitrenko@imbg.org.ua
Reverse transcriptase from avian myeloblastosis virus (AMV) was the subject of the study, from which the investi-
gations of the Department of biosynthesis of nucleic acids were started. Production of AMV in grams quantities
and isolation of AMV reverse transcriptase were established in the laboratory during the seventies of the past cen-
tury and this initiated research on the cDNA synthesis, cloning and investigation of the structure and functions of
the eukaryotic genes. Structures of salmon insulin and insulin-like growth factor (IGF) family genes and their
transcripts were determined during long-term investigations. Results of two modern techniques, microarray-ba-
sed hybridization and SAGE, were used for the identification of the genes differentially expressed in astrocytic
gliomas and human normal brain. Comparison of SAGE results on the genes overexpressed in glioblastoma with
the results of microarray analysis revealed a limited number of common genes. 105 differentially expressed genes,
common to both methods, can be included in the list of candidates for the molecular typing of glioblastoma. The
first experiments on the classification of glioblastomas based on the data of the 20 genes expression were conduc-
ted by using of artificial neural network analysis. The results of these experiments showed that the expression pro-
files of these genes in 224 glioblastoma samples and 74 normal brain samples could be according to the Koho-
nen’s maps. The CHI3L1 and CHI3L2 genes of chitinase-like cartilage protein were revealed among the most
overexpressed genes in glioblastoma, which could have prognostic and diagnostic potential. Results of in vitro
experiments demonstrated that both proteins, CHI3L1 and CHI3L2, may initiate the phosphorylation of ERK1/
ERK2 and AKT kinases leading to the activation of MAPK/ERK1/2 and PI3K/AKT signaling cascades in human
embryonic kidney 293 cells, human glioblastoma U87MG, and U373 cells. The new human cell line 293_CHI3L1,
stably producing chitinase-like protein CHI3L1 was developed and these cells were found to have an accelerated
growth rate and could undergo anchorage-independent growth in soft agar which is one of the most consistent
indicators of oncogenic transformation. The formation of tumors in rats by 293_CHI3L1 cells evidences that
CHI3L1 is an oncogene involved in tumorigenesis. In vitro experiments showed that constitutive expression of
CHI3L1 gene promotes chromosome instability in 293 cells.
Keywords: reverse transcriptase, brain tumors, differential gene expression, chitinase-like proteins, CHI3L1
oncogene.
Some history. The beginning of the investigations which
are carried out in the Department of biosynthesis of
nucleic acids may be related to the early 70th when the re-
action of reverse transcription was discovered [1, 2].
However, the scientists of the Institute of molecular bio-
logy and genetics were ready to accept this great disco-
very because already in 1961 Professor S. M. Gershen-
son hypothesized that the process of reverse transcrip-
tion might exist in living organisms [3]. Unfortunately,
at that time the Institute did not have any facilities to
conduct such extraordinary sophisticated experiments
in this field, so two scientists Alla Rynditch and Vadym
Kavsan began the first experiments on synthesis of
cDNA by AMV reverse transcriptase in the Institute of
molecular biology (Moscow), first in the lab of Dr. R.
Sh. Bibilashvili and then in the lab of Prof. V. A. Engel-
gardt. They were the first in the former Soviet Union,
who synthesized the globin cDNA in 1974 and later re-
verse transcribed the messenger RNAs of mouse plas-
mocytoma [4] and in such a way put the first brick in the
221
ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 3. P. 221–233 doi: 10.7124/bc.00081C
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013
222
organization of the International Project «Revertase-
oncogene», which was the beginning of genetic engi-
neering and biotechnology in the country. The main
participants of the Project got the State Prize in 1979.
After returning to the Institute in Kiev they organi-
zed the second in the world (after the Bird and Bird’s
laboratory in the United States) unique laboratory on
the production of avian myeloblastosis virus in grams
quantities and isolation of AMV reverse transcriptase
to supply it to many laboratories of different countries.
The possession of very substantial amounts of this uni-
que enzyme [5] allowed the scientists of the Institute to
have a wide collaboration in different fields of molecu-
lar biology. Thus, they tried to find reverse transcrip-
tion activity and discovered DNA-dependent DNA po-
lymerase activity associated with Galleria mellonella L.
nuclear polyhedrosis virus [6], performed comparative
study on RNA-dependent DNA-polymerases (reverse
transcriptases) of avian myeloblastosis and visna viru-
ses [7], and showed the sequence homology between
BSMV RNA species [8]. The development of new me-
thod for determination of poly(A)-sequences in RNAs
with the help of reverse transcription [9] gave the oppor-
tunity to detect the polyadenilate sequences in RNA
components of burley stripe mosaic virus [10].
We were the first who performed the DNA synthesis
by AMV DNA polymerase on the heterogeneous nuc-
lear RNA template [11] and on giant nuclear RNA [12].
In collaboration with Prof. G. P. Georgiev (Moscow) it
was discovered and confirmed on the original models
the ambiguous transcription boundaries in eukaryotes
that allowed detecting a mechanism of the processed
genes formation. It was shown for the first time that
cDNA molecule, synthesized on pre-mRNA template,
had a «lasso»-like structure [13–15] reflecting lasso-
like form of RNA in splicing that was confirmed later
by biochemical analyses. This finding was useful for the
understanding of the pre-mRNA splicing mechanisms
[16, 17].
Not only optimal conditions of the cDNA synthesis
by AMV reverse transcriptase was established during
the long period of the investigations [18–20] but AMV
reverse transcription was also used as a model for scree-
ning and study of chemotherapeutic agents against ret-
roviral infections, particularly 3'-asido-2',3'-dideoxy-
thymidine which later was employed for the HIV1 treat-
ment [21–23]. The new strain of HIV1 and the nucleo-
tide sequence of its genome has been firstly described
in Ukraine [24]. The structure of RSV adapted forms
was determined that allowed to elucidate the mecha-
nisms of the adaptation of oncogenic retroviruses to
new hosts and to show that for the maintenance of tumo-
rigenesis is not necessary to save a viral oncogene initia-
ted this process proving the invalidity of oncogene ad-
diction conception [25–28].
Several eukaryotic genes were synthesized by AMV
reverse transcriptase and their structures were analyzed
after cloning in plasmid vectors. Cloning of the rabbit
globin cDNA and analysis of the globin-specific sequ-
ences in poly(A)-containing pre-mRNA from rabbit bo-
ne marrow erythoroid cells allowed to characterize the
structure of globin-gene family as a model of eukaryotic
gene structure [29–32]. Scientists of the department par-
ticipated in the investigations on interferon genes and
the construction of recombinant plasmids pIFN-Ftrp
encoding synthesis of human leukocyte interferon cDNA
with the aim of obtaining recombinant protein as thera-
peutic agent, first in the former Soviet Union [33, 34].
Insulin and insulin-like growth factors genes. The
structures of salmon insulin-like growth factor I (IGF1)
and insulin-like growth factor II (IGF2) genes and their
promoter regions were determined after long-term in-
vestigations. Study on these genes, encoding growth-
promoting peptides, as started from isolation and deter-
mination of nucleotide sequences of cDNA clones from
salmon Brockman bodies cDNA library and as continu-
ed by the construction of salmon genome library and
examination of the corresponding genomic clones [35–
44]. Allelic polymorphism was described for insulin,
IGF1 and IGF2 genes during analysis of the salmon ge-
nome [45–47]. For the first time it was shown that the
salmon genome contained growth hormone pseudo-
gene [48] as well as two insulin genes [46] and two insu-
lin-like growth factor I genes; the second nonallelic
IGF1 gene was isolated from salmon genomic DNA lib-
rary [47]. Salmon IGF genes and their promoters were
investigated in details [38, 39]. It was revealed that the
chum salmon IGF1 promotor is activated by hepatocy-
te nuclear factor 1 [40]. At the same time it was shown
that IGF2 promoter was activated by hepatocyte nuc-
lear factor 3� [42] and requires Sp1 for its activation by
C/EBPb [43].
DMITRENKO V. V. ET AL.
Phylogenetic analysis of IGFs and their receptors
was carried out on the basis of the obtained results and
analysis of evolutionary conservation provided insights
into the essential regions of molecules of these hormo-
nes and their receptors [49].
Searching for new glioblastoma markers. Inves-
tigations of tissue-specific genes expression were carri-
ed out by analysis of human liver and brain cDNA libra-
ries at that time when Human Genome Project was star-
ted [50–53]. This study was transformed into investiga-
tions of the role of gene expression changes in the initia-
tion and progression of brain tumors. Several dozens of
genes differentially expressed in glioblastoma, the most
aggressive form of the brain tumors, and normal brain
were identified by differential hybridization of human
brain cDNA library [54–57]. To identify the genes that
might be used as molecular markers of glial tumors,
gene expression in astrocytic gliomas of WHO grade
II–IV and normal adult human brain was analyzed by
Serial Analysis of Gene Expression (SAGE) [58, 59]. In
our first work [58], the comparison of five glioblastoma
(GB) SAGE libraries with two normal brain (NB) SAGE
libraries, available at that time, has revealed 117 genes
with more than 5-fold difference of expression levels in
GB, P � 0.05.
Four new GB SAGE libraries appeared thereafter in
the SAGE Genie database and the amount of other ast-
rocytoma SAGE libraries were also increased. Thus, ni-
ne SAGE libraries of human glioblastoma (WHO grade
IV astrocytoma, GB), eleven SAGE libraries of human
anaplastic astrocytoma (WHO grade III, AA), eight
SAGE libraries of human astrocytoma (WHO grade II,
A) and five SAGE libraries of normal human brain (NB)
were analyzed to compare gene expression in astrocytic
gliomas with that of NB by accessing SAGE NCBI web
site (http://www.ncbi.nlm.nih.gov/SAGE) and using the
search tool of Digital Gene Expression Displayer
(DGED) provided by the SAGE Genie database [59].
Comparison of the pools of 9 GB SAGE libraries and 5
NB SAGE libraries has revealed 129 genes with more
than 5-fold differences in expression level compared to
NB. 44 genes of 129 met the criteria for genes overex-
pressed in tumors. The number of genes with more than
5-fold differences in AA was 66 genes with 18 genes as
overexpressed. 42 genes were shown as differentially
expressed in diffuse astrocytoma, 16 of them increased
their expression. Thus, the obtained results showed that
the number of genes activated in astrocytic tumors was
increasing during malignancy progression. Some ex-
pression changes occured early in astrocytoma forma-
tion and remained through passage to more malignant
state, other changes were characteristic only of the most
malignant stages of astrocytoma [59]. Northern blot
analysis confirmed SAGE results in several arbitrarily
selected differentially expressed transcripts. The expres-
sion patterns were usually reproducible between diffe-
rent samples. It is important to note, however, that there
were differences in the gene expression levels between
individual GBs [59]. Such differences in the gene ex-
pression undoubtedly contribute to the observed hetero-
geneity in the biological properties of cancers derived
from the same organ [60, 61].
To enhance glioblastoma marker discovery we used
DGED analysis of the pools of 9 GB SAGE-libraries
and 5 NB SAGE libraries and revealed 676 genes, 316
of which were determined as overexpressed. To compa-
re our SAGE results on the genes, which changed their
expression in GB with those obtained by microarray te-
chnique, the expression factor 2 and significance filter
P � 0.05 were chosen because these parameters were
used mostly in the microarray analyses. Unfortunately,
the comparison of even available data shows quite poor
overlapping of the genes revealed by microarrays in dif-
ferent papers. The explanation of such significant diffe-
rences in obtained results was given in four indepen-
dent studies [66–69], which confirmed three persistent
criticisms of the approach: the bewildering array of plat-
forms and research protocols available make results
from different studies hard to compare; in the hands of
less experienced labs, homemade arrays are less depen-
dable than commercial chips; different labs doing the
same study can often get very different results. Compa-
rison of our SAGE results on the genes overexpressed
in GB with microarray analysis results revealed a limi-
ted number of common genes. Alltogether, 105 of 849
described genes were overlapping with those obtained
by SAGE.
From our point of view, the main problem in evalua-
tion of results obtained by comparison of gene expres-
sion in glioblastomas and normal brain samples was the
lack of available data from each paper. The reason of
poor overlapping of the genes revealed by microarrays
223
FROM REVERSE TRANSCRIPTION TO HUMAN BRAIN TUMORS
apparently is due to methodological artefacts (e. g. dif-
ferent gene numbers placed onto chips, poor quality of
synthesized total cDNA probes or high background of
hybridization patterns, problems with house-keeping
gene controls, etc.) as well as to biological reasons (e. g.
heterogeneity of molecular mechanisms of glioblasto-
ma formation). A very big problem is obtaining normal
brain samples. Usually, surgical specimens of histologi-
cally normal brain, adjacent to the tumor, are used as the
source of normal brain RNA, however they can be consi-
dered as a normal control only with some precautions:
gliomas are infiltrating tumors and scattered tumor cells
are present far away from the dense tumor area removed
during surgery. Apparently, the best solution of the pro-
blem in searching for GB markers is to compare all avai-
lable results and to select only those genes, which signi-
ficant expression in the tumor combined with no detec-
table or very low expression in normal tissues was re-
produced in several articles. 105 differentially expres-
sed genes, revealed by both methods, can be included in
the list of candidates for the molecular typing of GB [62].
Some of overexpressed in glioblastoma genes may
encode oncoproteins and some underexpressed genes
may be the tumor suppressor genes. Thus, functional
analysis of several identified differentially expressed
genes was carried out to clarify the role of interaction of
potential oncoproteins and tumor suppressor proteins
with RAS/MAPK and PI3K/AKT signaling cascades,
involvement of these interactions in the malignant trans-
formation of brain cells, acquirement of proliferative
and invasive properties by tumor cells. Products of over-
expressed genes participate in angiogenesis, immunity,
ECM, cell signaling pathways, and related to the IGF-
system.
The genes of IGF-like family in glioblastoma. In
recent years, the evidences have appeared that the mem-
bers of IGF system may be involved in cancer develop-
ment. Increased expression of IGF1, IGF2, their recep-
tors, and binding proteins, or combinations thereof has
been documented in various malignancies including
gliomas. The results of multiple investigations suggest
that the IGFs can play a paracrine and/or autocrine role
in promoting tumor growth in situ during tumor pro-
gression but it may vary depending on the tissue of ori-
gin. Despite that the role of IGFs, IGF receptors, and
IGF-binding proteins (IGFBPs) in tumor development
is poorly understood up to this time, the antisense stra-
tegies, directed to the components of IGF-signaling, are
the subject of many clinical trials. All three IGF recep-
tors (IGF1R, INSR and IGF2R) are very well known
targets for anti-cancer therapy. The increased expres-
sion of IGF1 receptor as well as its ligands may stimula-
te the PI3K and MAPK signaling cascades leading to
cell proliferation.
We analyzed the expression of IGF system mem-
bers including all ten IGFBP genes in glioblastoma by
different methods to clarify their expression patterns in
this tumor However, enhanced expression of the IGF1
gene in glioblastomas was not found when we used
SAGE or analyzed data from the GEO repository [74].
Taking into account quite a big number of samples and
different methods used in the present investigation, the-
se results indicate that increased IGF1 gene expression
might be involved in the formation of only limited part
of astrocytic gliomas. Although the IGF1 was proposed
as one of targets for glial tumor therapy and was suppo-
sed to become the alternative treatment of human glio-
blastoma [75], our results clearly showed why the anti-
IGF1 treatment cannot give positive results with glio-
mas, supposing that the development of these tumors is
activated by some other way. In contrast to IGF1, the
expression of the IGF2 gene is up-regulated in glio-
blastoma. The microarray analysis data showed the exi-
stence of separate group of the glioblastomas overex-
pressing the IGF2 gene. This finding was in agreement
with the results of Soroceanu et al. [76] who found that
among 165 primary high-grade astrocytomas, 13 % of
glioblastomas and 2 % of anaplastic astrocytomas ex-
pressed IGF2 mRNA at the levels 50-fold higher than
sample population median. IGF2 can substitute for EGF
to support the growth of glioblastoma-derived neuro-
spheres and growth-promoting effects of IGF2 were
mediated by the IGF1 receptor and phosphoinositide-
3-kinase regulatory subunit 3 (PIK3R3), a regulatory
subunit of PI3K.
The results of the analysis of IGFBP genes expres-
sion in glioblastoma, obtained by three methods (SAGE,
microarray analysis and RT-PCR), demonstrate up-re-
gulation of the majority IGFBPs in this tumor. Pro-
duced in tumor cells, IGFBPs may stabilize insulin-like
growth factor(s), IGF1 and/or IGF2, and drive their ac-
tivation in glial tumors [74]. On the other hand, some of
224
DMITRENKO V. V. ET AL.
the IGFBPs inhibit IGF actions or may act by a mecha-
nism independent of IGFs, as reviewed by Mohan and
Baylink [77].
Thus, increased expression of the IGF-binding pro-
tein genes in brain tumors makes the picture even more
complicated. Our data highlight the importance of view-
ing the IGF-related proteins as a complex multifactorial
system and show that changes in the expression levels of
any one component of the system, in a given malignan-
cy, should be interpreted with caution. As IGF targeting
in anti-cancer therapy is rapidly becoming clinical reali-
ty, an understanding of this complexity is very timely.
The chitinase-like genes in glioblastoma. As it was
described above, IGF1 is a key peptide in many tumors
but was not found as overexpressed in glioblastoma
[74], so it was supposed that IGF1 participation in the
development of glial tumors may be substituted by pro-
tein products of other highly expressed genes, also par-
ticipating in the PI3K and MAPK pathways. Chitina-
se-like glicoprotein CHI3L1 (other names YKL-40 and
HC-gp39), encoding by the CHI3L1 gene with conside-
rably increased expression in most part of glioblasto-
mas [78] could participate instead of IGF1 in the deve-
lopment of glioblastoma formation. It was shown that
just as IGF1, it may stimulate the ERK1/2- and AKT-
signaling pathways, associated with mitogenesis cont-
rol, in a concentration range similar to the effective dose
of IGF1 [79]. The new human cell line 293_CHI3L1
stably producing CHI3L1 was constructed and found
that these cells had an accelerated growth rate and could
undergo anchorage-independent growth in soft agar
what is one of the most consistent indicators of oncoge-
nic transformation [80, 81]. 293_CHI3L1 cells had acti-
vated PI3K and MAPK pathways; phosphorylated pro-
tein kinase B (AKT) was localized in cytoplasm while
extracellular signal regulated kinases (ERK1/2) were
localized in both cytoplasm and nuclei where they
could activate different transcription factors with cer-
tain biological outcome. The CHI3L1 gene knockdown
by siRNA transfection gave noticeable CHI3L1 protein
blockade (80–90 %) with significantly reduced pERK1/
2 and the colony-forming ability in soft agar of 293_
CHI3L1 cells. The formation of tumors in rats by 293
cells expressing CHI3L1 evidences that CHI3L1 is an
oncogene which is involved in tumorigenesis. This was
the first animal model of human brain tumor which
could be used for studying various biological proper-
ties of brain tumors in the immunocompetent animals
[82]. The obtained results demonstrate that the activity
of CHI3L1 mediated by pathways involved ERK1/2 and
AKT plays a growth-promoting role and the overex-
pression of CHI3L1 is likely to be critical in the develop-
ment of some tumors.
Other gene with considerably increased expression
in glioblastoma identified by SAGE was CHI3L2 (YKL-
39) encoded 39 kDa chitinase-like protein that like
CHI3L1 is a member of the 18 glycosyl hydrolase fami-
ly [83]. Northern blot hybridization confirmed the data
of SAGE for the majority of glioblastomas [84]. High
homology of nucleotide and amino acid sequences of
CHI3L2 and CHI3L1 suppose some identity of their
functions [85]. However, western blot analysis did not
show simultaneous production of CHI3L2 and CHI3L1
in glioblastoma and anaplastic astrocytoma samples
that evidence the differences in functions of these ho-
mologous proteins [84]. CHI3L2 also induced phospho-
rylation of ERK1/ERK2 as CHI3L1 did. The results of
in vitro experiments demonstrated that both proteins,
CHI3L1 and CHI3L2, might initiate the phosphoryla-
tion of ERK1/ERK2 leading to the initiation of MAP
kinase signaling cascade in human embryonic kidney
293 cells, human glioblastoma U87MG, and U373 cells
[86, 87]. Activation of ERK1/2 by CHI3L2 was more
prolong than by CHI3L1, and declined after 2 h only by
~ 30 % while after activation by CHI3L1 it declined ap-
proximately to a basal level. In contrast to the activa-
tion of ERK1/2 phosphorylation by CHI3L1 that lead
to a proliferative signal (similar to the EGF effect in
PC12 cells), activation of ERK1/2 phosphorylation by
CHI3L2 (similar to NGF) inhibited cell mitogenesis and
proliferation. The diversity in their functional activities
could be explained, firstly, by the fact that native CHI3L1
is glycosylated at Asn60 while CHI3L2 is not a glyco-
protein. Besides, CHI3L1 has a cluster of basic residues
which can bind heparin; CHI3L2 has a different amino
acid sequence in this site. Third, in the ligand-binding
groove CHI3L1 has two tryptophan residues, in CHI3L2
these tryptophans are mutated to lysines which change
the protein charge and hydrophobicity [88].
Growing body of evidence suggests that sustained
activation and nuclear migration of AKT is implicated in
controlling of differentiation and apoptosis in several
225
FROM REVERSE TRANSCRIPTION TO HUMAN BRAIN TUMORS
cell lines [89, 90]. The involvement of CHI3L1 and
CHI3L2 proteins in activation of PI3K/AKT pathway
and treatment of 293 cells by CHI3L2 resulted in fast
increase of AKT phosphorylation, which continued for
a long time. Similar sustained phosphorylation was ob-
served also for U373 cells. The obtained data demonst-
rated prolonged activation of AKT by the CHI3L2 pro-
tein like to previously reported data for NGF in PC12
cells [89, 91]. In contrast to CHI3L2, CHI3L1-induced
time course activation of AKT demonstrated substantial-
ly different result – incubation of the cells with CHI3L1
caused more transient activation of AKT. The phospho-
rylated form of AKT after incubation with CHI3L2 pro-
tein was detectable both in cytoplasm and nucleus as
opposed to localization of phosphorylated AKT under
CHI3L1 influence in the cytoplasm only. Overall, the
results of AKT induction by CHI3L1 and CHI3L2 sug-
gest that CHI3L1 potentially has a significantly diffe-
rent effect on the function of this kinase as compared to
CHI3L2. The cellular receptors mediating the biolo-
gical effects of CHI3L1 and CHI3L2 are not yet known
but the activation of cytoplasmic signal-transduction
pathways suggests that these chitinase-like proteins in-
teract with one or several signaling components on the
plasma membrane. Different results of these interactions,
revealed for CHI3L1 and CHI3L2, may have various
impacts on the fate of the cells.
Glioblastoma treatment. Despite revealed in vitro
anti-proliferative effect of the CHI3L1 oncoprotein sup-
pression, therapy against one oncogene target cannot be
effective and in the present decade it is believed that
cancer therapy is going to shift slowly from «one tar-
get» to a more personalized multitarget approach. High
heterogeneity of glial tumors makes necessary simulta-
neous analyses of many genes and therapy targeted not
to individual genes, but to the physiological effect cau-
sed by these genes. Angiogenesis is an important part
of the tumor development, through which the nutrients
get into the centre of tumor developed in hypoxic con-
ditions. This allows the cells of malignant neoplasms to
proliferate under increased oxygenation conditions and
removal of metabolic wastes which usually induce nec-
rosis. When the number of tumor cells reaches a critical
level, they contribute to the formation of new blood ves-
sels and metastasis. A number of angiogenic factors
such as VEGF and PDGF, play a key role in tumor vas-
cularization. In anticancer therapy, a considerable at-
tention paid to the anti-angiogenic drugs, which are
approved by FDA, such as bevacizumab (antibodies
against VEGF) and tyrosine kinase inhibitors of VEGF
receptor (sorafenib and sunitinib). However, the success
of these angiogenic drugs is a temporary one, the drug
resistance, tumor recurrence and rapid firmation of the
new blood vessels are developed at the end of the thera-
py. Besides, the opposite effect of angiogenic agents on
tumor growth takes place, as well as on the angioge-
nesis and metastasis formation in xenograft tumor mo-
dels as well. Oncogenic redundancy is a significant ob-
stacle to the success of against targeting treatment. It
was shown that CHI3L1 possessed highly proangio-
genic properties [92]. So, simultaneous treatment with
anti-CHI3L1 (as specific siRNAs) and anti-VEGF (as
Bevacizumab) preparations may give positive results.
Of course, an obvious success may be predicted for
complex cancer therapy with chemical preparations of
different types. The study was initiated by evaluation of
anticancer activity in compounds with distinct chemi-
cal nature, namely bradykinin (BK) antagonists and azo-
lidinones-related chemicals, in several types of malig-
nantly transformed cells: 293 cells, stably transfected
by CHI3L1 oncogene (293_CHI3L1), glioblastoma-
derived U373 cells and mantle cell lymphoma (MCL)
cell lines Granta, JeKo, Mino and UPN1. Nonapeptides
BK possess many different activities related to normal
physiology as well as to pathophysiology, namely the
modulation of vascular tone, pain, and inflammation.
BK has been shown to have growth-factor properties in
human cancers of lung, prostate, ovarian, gastrointes-
tinal, and breast, promotes the migration of glioma cells
[93]. This formed the basis for development of new drugs,
such as BK antagonists. We showed that several brady-
kinin antagonists have significant growth suppressor
activity in 293_CHI3L1 and U373 cells and strongly in-
hibited extracellular signal-regulated kinases 1/2 (ERK1/
2) and protein kinase B (AKT1) phosphorylation. Azo-
lidinones are of great importance in modern medicinal
chemistry and have been investigated for a range of
pharmacological activities such as anti-inflammatory,
antimicrobial, antiviral, antiproliferative, etc. Special
attention was attracted to azolidinones as potential no-
vel anticancer agents. Previously, the group of Dr. Ro-
man Lesyk at Danylo Halytsky Lviv National Medical
226
DMITRENKO V. V. ET AL.
University reported about growth-suppression activity
of azolidinone derivatives, particularly against gliobla-
stoma cell lines [94]. We found that one of examined
preparations demonstrated high anti-proliferative pro-
perties. Thus, a growth suppression activity of two dif-
ferent classes of molecules was shown in three types of
malignantly transformed cells. Our preliminary results
demonstrated that molecular mechanisms of their ac-
tion might rely on the modulation of key cellular sig-
naling pathways. Further investigations of molecular
mechanisms of BK antagonists and azolidinone deriva-
tives action and pre-clinical studies using animal mo-
dels are needed for the evaluation of these compounds
as new anti-cancer drugs.
The description of new molecular markers is neces-
sary for identification of specific gene expression profi-
les (signatures) in tumor cells which will be useful for
understanding the molecular mechanisms participating
in the arising and development of neoplasms as well as
for determining the strategy of anticancer therapy. We
made first attempts to develop such signature for gliobla-
stoma. Neural network analysis was used for the classi-
fication of glioblastomas based on the data of 20 diffe-
rentially expressed genes revealed by microarray analy-
sis. The obtained results showed that the expression pro-
files of these genes in 224 glioblastoma samples and 74
normal brain samples can be clustered using the Koho-
nen’s maps [95]. Subsequent comparison of the micro-
array analysis and SAGE results showed that about 30
differentially expressed genes are suitable for the recog-
nition of specific gene expression profiles in gliobla-
stomas and normal brain by artificial neural network.
For drugs delivery into the brain we develop the na-
nocojugates which should penetrate across the blood-
brain and tumor-normal brain barriers. A therapeutic
agent of created nanoconjugates is a morpholino anti-
sense-oligonucleotide or siRNA to CHI3L1 mRNA and
some other agents. As a vector is used a polymer matrix
presenting natural biopolymer poly-beta-maleic acid
(�-L-malic acid, PMLA) from the microorganism Phy-
sarum polycephalum, which was isolated and transfer
red to us by Dr. J. Lyubimova (Cedars-Sinai Medical
Center, USA). Previously, the polyfunctional nano-
conjugate Polycefin was developed in Dr. J. Ljubimova
lab (Polycefin, US patent 2007/0259008 A1) and its in-
hibitory effect was demonstrated on the tumor growth
in the brain of «nude» rats by inhibition of laminin-8
vascular protein overproduction in glioblastoma cells
[96]. In our experiments, antisense-oligonucleotide to
the CHI3L1 mRNA will be attached to PMLA polyme-
ric matrix via disulphide bonds which have to be dis-
rupted in cytoplasm to release therapeutic agent(s) wi-
thin cells. In addition to the therapeutic agent, several
modules have been introduced into nanoconjugates re-
quired for directed delivery to the tumor cells as poly-
ethylene glycol (PEG) to protect conjugate against ra-
pid degradation, trileucine peptide for pH-dependent li-
pophilicity provision to destruct the endosomal memb-
ranes, antibodies against transferrin receptor (TfR) for
getting into tumor cells and receptor-mediated endocy-
tosis, reporter fluorescent dye for therapeutic detection (if
necessary). It is expected that nanoconjugates of antisen-
se-oligonucleotides or siRNA to CHI3L1 mRNA with
PMLA will have antiproliferative effect and will inhi-
bit tumor cells growth due to suppression of CHI3L1
protein production in. Developed by us the new model
of brain tumor in immunocompetent adult rats will be
used to test these nanocojugates in vivo [82].
Cancer cells karyotyping and evolution of can-
cer. Chromosome instability (CIN) and the resulting
clonal/non-clonal intratumor heterogeneity elucidate
why large-scale tumor genome sequencing and high-re-
solution analysis of somatic copy-number alterations
have failed to reveal «universal» cancer genes except
well known for decades, and type- and stage-specific
recurrent aberrations in solid tumors, whereas most re-
current chromosome aberrations (deletions, amplifica-
tions, and translocations) ever occurring genome-wide
in tumors can be explained by 3D genome organiza-
tion, spatial proximity among chromosome loci, and
replication timing of sites producing rearrangements.
CIN explains how mutagenic and non-mutagenic che-
mical agents, physical factors, contacts with bacterial
cells, and infection with some viruses induce or promo-
te transformation of cells in vitro and tumor develop-
ment in vivo, as well as spontaneous in vitro transfor-
mation of primary and immortalized cells and tumori-
genicity of induced pluripotent stem (iPS) cells. CIN ac-
counts for the acquisition of oncogene independence
and tumor recurrence after inductor withdrawal in onco-
gene on/off conditional transgenic mice models. CIN
and intratumor heterogeneity are the reasons of onco-
227
FROM REVERSE TRANSCRIPTION TO HUMAN BRAIN TUMORS
gene addiction independence of solid tumors from any
particular oncogene and general ineffectiveness of tar-
geted therapy in clinic. Any factors or stresses that cont-
ribute to CIN can potentially promote the evolution of
cancer (reviewed in [97]).
The process of cellular transformation has been
amply studied in vitro using immortalized cell lines. Im-
mortalized cells never have the normal diploid karyo-
type, nevertheless, they cannot grow over one another
in cell culture (contact inhibition), do not form colonies
in soft agar (anchorage-dependent growth) and do not
form tumors when injected into immunodeficient ro-
dents. All these characteristics can be obtained with ad-
ditional chromosome changes. Multiple genetic rearran-
gements, including whole chromosome and gene copy
number gains and losses, chromosome translocations,
gene mutations are necessary for establishing the malig-
nant cell phenotype. Most of the experiments detecting
transforming ability of genes overexpressed and/or mu-
tated in tumors (oncogenes) were performed using mou-
se embryonic fibroblasts (MEFs), NIH3T3 mouse fibro-
blast cell line, human embryonic kidney 293 cell line
(HEK293), and human mammary epithelial cell lines
(mainly HMECs and MCF10A). These cell lines have
abnormal karyotypes and are prone to progress to malig-
nantly transformed cells. The mechanisms of cell immor-
talization 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 chromoso-
me addiction», intra/intertumor heterogeneity, and chro-
mosome instability are just discussed in review [98]).
For decades the conventional gene mutation cancer
theory has been postulating that cancer is a genetic di-
sease considered as a result of deterministic sequential
accumulation of the mutations in handful of «driver»
cancer genes occurring in a continuous linear pattern of
cancer progression. However, in contrast to this postu-
late, the recent whole genome and exome sequencing
studies of primary tumor bulk and metastases or sepa-
rate regions within the same sample have revealed a lar-
ge number of stochastic gene mutations for each indivi-
dual with the same cancer type and significant intratu-
moral genetic heterogeneity with «branched evolutio-
nary tumor growth» or «punctuated clonal evolution wi-
thout observable intermediate branching» or «no domi-
nant clones in the cancer tissue». Meanwhile, the sto-
chastic karyotypic variation and intratumor heteroge-
neity are recognized to be the driving force of tumor
evolution and major factors of recurrent tumors occur-
rence with acquired drug resistance. The karyotype evo-
lution/chromosome instability and the resulting magni-
tude of intratumor heterogeneity significantly correlate
with tumorigenic potential of cells, tumor disease pro-
gression from precancerous lesions to malignant tumors
and metastases, correlate with patient survival, treat-
ment sensitivity, and the risk of acquired resistance. We
discuss importance of the evolutionary karyotypic theo-
ry in understanding of the cancer biology and mecha-
nisms of tumor drug resistance [99].
Recently we have revealed that constitutive expres-
sion of CHI3L1 promotes chromosome instability in 293
cells. Modal number of chromosomes in 293_CHI3L1
cells is distinct to that in transfection control 293_
pcDNA3.1 cells and parental 293 cells. Interline whole
chromosome heterogeneity is manifested. A number
of new distinct marker chromosomes were observed in
CHI3L1-expressing cells from two independent experi-
ments. Array comparative genome hybridization (aCGH)
was used to analyze the subchromosomal alterations in
these cell lines. The spectrum of cytoband gains and los-
ses in 293_CHI3L1 cells was significantly different
from control cells. Thus, we established the link between
transforming properties of oncogene CHI3L1 and chan-
ges of karyotype of 293 cells with stable expression of
CHI3L1.
Conclusions. Reverse transcription and correspon-
ding enzymes played a key role in the development of
the investigations in the Department of biosynthesis of
nucleic acids (IMBG, NANU). Similar projects beca-
me the most rapidly growing fields in molecular biolo-
gy of 70–80 years of the last century. The possession of
very substantial amounts of this unique enzyme gave
opportunity for the fast creating of cDNA libraries on
mRNAs of different origin, isolation of respective cDNA
clones, determination of primary structure of different
eukaryotic genes, and study on the structure of corres-
ponding genome loci. Determined structures of the sal-
mon insulin and insulin-like growth factor (IGF) fami-
ly genes, their allelic polymorphism, promoters, and
their transcripts became classical in the investigations of
non-mammalian genomes.
228
DMITRENKO V. V. ET AL.
The most suitable modern methods for determi-
nation of genes differentially expressed in astrocytic
gliomas and human normal brain, SAGE and microar-
ray hybridization analysis based on cDNA synthesis
were used for identification of new glioma markers.
Differentially expressed genes, common to both me-
thods are the candidates for the molecular typing of glio-
blastoma.
It was found that overexpressed in glioblastomas the
CHI3L1 gene encoding chitinase-like protein CHI3L1
had oncogenic properties. Strategies, based on the comp-
lex therapy including inhibition of CHI3L1 expression
by nanocojugates of Morpholino antisense oligonucleo-
tide to the CHI3L1 mRNA and polymalic acid, will be
used for the developing of the brain tumors therapy.
In vitro experiments showed that the constitutive ex-
pression of CHI3L1 gene promotes chromosome insta-
bility in 293 cells. Modal number of chromosomes in
293_CHI3L1 cells differs from that in transfection
control 293_pcDNA3.1 cells and parental 293 cells. A
number of new distinct marker chromosomes were ob-
served in CHI3L1-expressing cells from two indepen-
dent experiments. Thus, the link between transforming
properties of oncogene CHI3L1 and changes in karyo-
type of 293 cells, stably producing CHI3L1 protein,
was established.
Acknowledgements. This research was supported
by National Academy of Sciences of Ukraine in frames
of the program «Fundamental grounds of molecular
and cell biotechnologies», Project «New molecular and
genetic markers for gene expression signatures of brain
tumors and their interactions with signaling pathways»,
program «Nanotechnologies and nanomaterials for
2010–2014 years», Project «Formation of brain tumor
growth inhibition system based on nanoconjgates of
antisense oligonucleotides, specific to oncoprotein
mRNAs, with natural biopolymers»; in frames of joint
program between NAS of Ukraine and the Russian Fund
of Fundamental Researches in 2012, Project 07-04-12
(Ó) and by Science and Technology Center in Ukraine,
project 5446 «Nanoconjugates of natural biopolymers
with antisense oligonucleotides and antibody for inhibi-
tion of glial tumors» and by State Agency for Science,
Innovations and Informatization of Ukraine in frames
of the project F46.2/01 «State Key Laboratory of Mole-
cular and Cell Biology».
Â. Â. Äìèòðåíêî, Ñ. Ñ. Àâ人â, Ï. Î. Àðåøêîâ, Î. Â. Áàëèíñüêà,
Ò. Â. Áóêðåºâà, Î. À. Ñòåïàíåíêî, Ò. É. ×àóñîâñüêèé, Â. Ì. Êàâñàí
³ä çâîðîòíî¿ òðàíñêðèïö³¿ äî ïóõëèí ãîëîâíîãî ìîçêó ëþäèíè
Ðåçþìå
Íàóêîâ³ ðîçðîáêè â³ää³ëó á³îñèíòåçó íóêëå¿íîâèõ êèñëîò ðîçïî÷à-
òî ç âèâ÷åííÿ çâîðîòíî¿ òðàíñêðèïòàçè â³ðóñó ïòàøèíîãî 쳺ëî-
áëàñòîçó (AMV). Ïðîòÿãîì ñ³ìäåñÿòèõ ðîê³â ìèíóëîãî ñòîë³òòÿ
ó â³ää³ë³ íàëàãîäæåíî âèðîáíèöòâî AMV (äåê³ëüêà ãðàì³â íà ð³ê)
òà âèä³ëåííÿ çâîðîòíî¿ òðàíñêðèïòàçè AMV, ùî äîçâîëèëî ðîç-
ãîðíóòè ðîáîòè ç ñèíòåçó êÄÍÊ, êëîíóâàííÿ òà âèâ÷åííÿ ñòðóê-
òóðè ³ ôóíêö³¿ ãåí³â åâêàð³îò³â. Óïðîäîâæ áàãàòîð³÷íèõ äîñë³ä-
æåíü áóëî âèçíà÷åíî áóäîâó ãåí³â ³íñóë³íó ³ ðîäèíè ³íñóë³íîïîä³á-
íèõ ôàêòîð³â ðîñòó (IGF) ëîñîñÿ òà ¿õí³õ òðàíñêðèïò³â. Ðåçóëü-
òàòè çàñòîñóâàííÿ äâîõ ñó÷àñíèõ ìåòîä³â – ã³áðèäèçàö³¿ ì³êðî÷³-
ï³â ³ SAGE – âèêîðèñòàíî äëÿ ³äåíòèô³êàö³¿ ãåí³â, ÿê³ äèôåðåí-
ö³éíî åêñïðåñóþòüñÿ â àñòðîöèòàðíèõ ãë³îìàõ ³ íîðìàëüíîìó ãî-
ëîâíîìó ìîçêó ëþäèíè. ¯õíº ïîð³âíÿííÿ âèÿâèëî îáìåæåíó ê³ëüê³ñòü
ñï³ëüíèõ ãåí³â, íàäåêñïðåñîâàíèõ ó ãë³îáëàñòîì³. Âèçíà÷åí³ íàìè
105 äèôåðåíö³éíî åêñïðåñîâàíèõ ãåí³â, ñï³ëüíèõ äëÿ îáîõ ìåòîä³â,
ìîæóòü áóòè âêëþ÷åí³ äî ïåðåë³êó êàíäèäàò³â äëÿ ìîëåêóëÿðíî-
ãî òèïóâàííÿ ãë³îáëàñòîì. Ïðîâåäåíî ïåðø³ åêñïåðèìåíòè ç êëà-
ñèô³êàö³¿ ãë³îáëàñòîì íà îñíîâ³ äàíèõ ïî åêñïðåñ³¿ 20 ãåí³â ³ç çà-
ñòîñóâàííÿì øòó÷íî¿ íåéðîííî¿ ìåðåæ³, ÿê³ ïîêàçàëè, ùî ïðîô³-
ë³ åêñïðåñ³¿ çàçíà÷åíèõ ãåí³â äëÿ 224 çðàçê³â ãë³îáëàñòîì ³ 74 çðàç-
ê³â íîðìàëüíîãî ãîëîâíîãî ìîçêó ï³ääàþòüñÿ êëàñòåðèçàö³¿ çã³äíî
ç êàðòàìè Êîõîíåíà. Ñåðåä íàéåêñïðåñîâàí³øèõ ó ãë³îáëàñòîì³ ãå-
í³â, ÿê³ ìàþòü ïðîãíîñòè÷íèé ³ ä³àãíîñòè÷íèé ïîòåíö³àë, âèÿâëå-
íî ãåíè õ³òèíàçîïîä³áíèõ á³ëê³â CHI3L1 ³ CHI3L2. Ðåçóëüòàòè åêñ-
ïåðèìåíò³â in vitro ïðîäåìîíñòðóâàëè, ùî îáèäâà á³ëêè – CHI3L1
³ CHI3L2 – çäàòí³ ³í³ö³þâàòè ôîñôîðèëþâàííÿ ê³íàç ERK1/ERK2
³ AKT, ùî ñïðè÷èíÿº àêòèâàö³þ ñèãíàëüíèõ êàñêàä³â PI3K/AKT ³
MAPK/ERK1/2 â êë³òèíàõ 293 åìáð³îíàëüíî¿ íèðêè ëþäèíè, à òà-
êîæ ó êë³òèíàõ U87MG ³ U373 ãë³îáëàñòîìè ëþäèíè. ²äåíòèô³êî-
âàíî íîâó êë³òèííó ë³í³þ ëþäèíè 293_CHI3L1, ÿêà ñòàá³ëüíî ïðî-
äóêóº õ³òèíàçîïîä³áíèé á³ëîê CHI3L1. Çíàéäåíî, ùî ö³ êë³òèíè ìà-
þòü ïðèñêîðåíèé ð³ñò ³ ìîæóòü ðîñòè ó ì’ÿêîìó àãàð³ íåçàëåæ-
íî â³ä ïðèêð³ïëåííÿ äî ïîâåðõí³, ùî º îäíèì ³ç íàéñóòòºâ³øèõ ïî-
êàçíèê³â ïóõëèííî¿ òðàíñôîðìàö³¿. Ôîðìóâàííÿ ïóõëèí êë³òèíà-
ìè 293_CHI3L1 ó ùóð³â ñâ³ä÷èòü ïðî òå, ùî CHI3L1 º îíêîãåíîì,
ïðè÷åòíèì äî êàíöåðîãåíåçó. Åêñïåðèìåíòè in vitro çàñâ³ä÷èëè,
ùî êîíñòèòóòèâíà åêñïðåñ³ÿ ãåíà CHI3L1 ñïðèÿº õðîìîñîìí³é íå-
ñòàá³ëüíîñò³ ó êë³òèíàõ 293. Ìîäàëüíå ÷èñëî õðîìîñîì ó êë³òè-
íàõ 293_CHI3L1 â³äð³çíÿºòüñÿ â³ä òàêîãî õðîìîñîì ó êîíòðîëü-
íèõ êë³òèíàõ 293_pcDNA3.1, òðàíñô³êîâàíèõ «ïîðîæí³ì» ïëàç-
ì³äíèì âåêòîðîì, ³ áàòüê³âñüêèõ êë³òèíàõ 293.
Êëþ÷îâ³ ñëîâà: çâîðîòíà òðàíñêðèïòàçà, ïóõëèíè ãîëîâíîãî
ìîçêó, äèôåðåíö³éíà åêñïðåñèÿ ãåí³â, õ³òèíàçîïîä³áí³ á³ëêè, îíêî-
ãåí CHI3L1.
Â. Â. Äìèòðåíêî, Ñ. Ñ. Àâäååâ, Ï. À. Àðåøêîâ, Î. Â. Áàëûíñêàÿ,
Ò. Â. Áóêðååâà, À. À. Ñòåïàíåíêî, Ò. È. ×àóñîâñêèé, Â. Ì. Êàâñàí
Îò îáðàòíîé òðàíñêðèïöèè ê îïóõîëÿì ãîëîâíîãî ìîçãà ÷åëîâåêà
Ðåçþìå
Íàó÷íûå ðàçðàáîòêè îòäåëà áèîñèíòåçà íóêëåèíîâûõ êèñëîò íà-
÷àëèñü ñ èçó÷åíèÿ îáðàòíîé òðàíñêðèïòàçû âèðóñà ïòè÷üåãî ìè-
åëîáëàñòîçà (AMV).  òå÷åíèå ñåìèäåñÿòûõ ãîäîâ ïðîøëîãî âåêà
â îòäåëå íàëàæåíî ïðîèçâîäñòâî AMV (íåñêîëüêî ãðàììîâ â ãîä)
229
FROM REVERSE TRANSCRIPTION TO HUMAN BRAIN TUMORS
è âûäåëåíèå îáðàòíîé òðàíñêðèïòàçû AMV, ÷òî ïîçâîëèëî ðàç-
âåðíóòü ðàáîòû ïî ñèíòåçó êÄÍÊ, êëîíèðîâàíèþ è èññëåäîâàíèå
ñòðóêòóðû è ôóíêöèè ãåíîâ ýóêàðèîòîâ. Íà ïðîòÿæåíèè ìíîãî-
ëåòíèõ èññëåäîâàíèé áûëî îïðåäåëåíî ñòðîåíèå ãåíîâ èíñóëèíà è
ñåìåéñòâà èíñóëèíîïîäîáíûõ ôàêòîðîâ ðîñòà (IGF) ëîñîñÿ è èõ
òðàíñêðèïòîâ. Ðåçóëüòàòû ïðèìåíåíèÿ äâóõ ñîâðåìåííûõ ìåòî-
äîâ – ãèáðèäèçàöèè ìèêðî÷èïîâ è SAGE – èñïîëüçîâàíû äëÿ èäåí-
òèôèêàöèè ãåíîâ, äèôôåðåíöèàëüíî ýêñïðåññèðóþòñÿ â àñòðîöè-
òàðíûõ ãëèîìàõ è íîðìàëüíîì ãîëîâíîì ìîçãå ÷åëîâåêà. Èõ ñðàâ-
íåíèå âûÿâèëî îãðàíè÷åííîå ÷èñëî îáùèõ ãåíîâ, íàäýêñïðåññèðî-
âàííûõ â ãëèîáëàñòîìå. Îïðåäåëåííûå íàìè 105 äèôôåðåíöèàëüíî
ýêñïðåññèðîâàííûõ ãåíîâ, îáùèõ äëÿ îáîèõ ìåòîäîâ, ìîãóò áûòü
âêëþ÷åíû â ñïèñîê êàíäèäàòîâ äëÿ ìîëåêóëÿðíîãî òèïèðîâàíèÿ
ãëèîáëàñòîì. Ïðîâåäåíû ïåðâûå ýêñïåðèìåíòû ïî êëàññèôèêà-
öèè ãëèîáëàñòîì íà îñíîâå äàííûõ ïî ýêñïðåññèè 20 ãåíîâ ñ èñ-
ïîëüçîâàíèåì èñêóññòâåííîé íåéðîííîé ñåòè, ïîêàçàâøèå, ÷òî
ïðîôèëè ýêñïðåññèè ýòèõ ãåíîâ äëÿ 224 îáðàçöîâ ãëèîáëàñòîì è
74 îáðàçöîâ íîðìàëüíîãî ãîëîâíîãî ìîçãà ìîãóò áûòü êëàñòåðè-
çîâàíû â ñîîòâåòñòâèè ñ êàðòàìè Êîõîíåíà. Ñðåäè íàèáîëåå
íàäýêñïðåñèðîâàííûõ â ãëèîáëàñòîìå ãåíîâ, èìåþùèõ ïðîãíîñòè-
÷åñêèé è äèàãíîñòè÷åñêèé ïîòåíöèàë, îáíàðóæåíû ãåíû õèòèíà-
çîïîäîáíûõ áåëêîâ CHI3L1 è CHI3L2. Ðåçóëüòàòû ýêñïåðèìåí-
òîâ in vitro ïðîäåìîíñòðèðîâàëè, ÷òî îáà áåëêà – CHI3L1 è
CHI3L2 – ìîãóò èíèöèèðîâàòü ôîñôîðèëèðîâàíèå êèíàç ERK1/
ERK2 è AKT, ïðèâîäÿùåå ê àêòèâàöèè ñèãíàëüíûõ êàñêàäîâ PI3K/
AKT è MAPK/ERK1/2 â êëåòêàõ 293 ýìáðèîíàëüíîé ïî÷êè ÷åëîâå-
êà, à òàêæå â êëåòêàõ U87MG è U373 ãëèîáëàñòîìû ÷åëîâåêà.
Ïîëó÷åíà íîâàÿ êëåòî÷íàÿ ëèíèÿ ÷åëîâåêà 293_CHI3L1 ñî ñòà-
áèëüíîé ïðîäóêöèåé õèòèíàçîïîäèáíîãî áåëêà CHI3L1. Îáíàðó-
æåíî òàêæå, ÷òî ýòè êëåòêè îáëàäàþò óñêîðåííûì ðîñòîì è
ìîãóò ðàñòè â ìÿãêîì àãàðå íåçàâèñèìî îò ïðèêðåïëåíèÿ ê ïîâåðõ-
íîñòè. Òàêèå ñâîéñòâà ÿâëÿþòñÿ îäíèì èç íàèáîëåå ñóùåñòâåí-
íûõ ïîêàçàòåëåé îïóõîëåâîé òðàíñôîðìàöèè. Ôîðìèðîâàíèå îïó-
õîëåé êëåòêàìè 293_CHI3L1 ó êðûñ ñâèäåòåëüñòâóåò î òîì, ÷òî
CHI3L1 ÿâëÿåòñÿ îíêîãåíîì, ó÷àñòâóþùèì â êàíöåðîãåíåçå. Ýêñ-
ïåðèìåíòû in vitro ïîêàçàëè, ÷òî êîíñòèòóòèâíàÿ ýêñïðåññèÿ ãå-
íà CHI3L1 ñïîñîáñòâóåò õðîìîñîìíîé íåñòàáèëüíîñòè â êëåò-
êàõ 293. Ìîäàëüíîå ÷èñëî õðîìîñîì â êëåòêàõ 293_CHI3L1 îòëè-
÷àåòñÿ îò òàêîâîãî õðîìîñîì â êîíòðîëüíûõ êëåòêàõ 293_
pcDNA3.1, òðàíñôåöèðîâàííûõ «ïóñòûì» ïëàçìèäíûì âåêòî-
ðîì, è ðîäèòåëüñêèõ êëåòêàõ 293.
Êëþ÷åâûå ñëîâà: îáðàòíàÿ òðàíñêðèïòàçà, îïóõîëè ãîëîâíîãî
ìîçãà, äèôôåðåíöèàëüíàÿ ýêñïðåññèÿ ãåíîâ, õèòèíàçîïîäîáíûå
áåëêè, îíêîãåí CHI3L1.
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Received 30.12.12
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