Expression profiling of cyclin B1 and D1 in cervical carcinoma
Aim: Cyclins are a family of regulatory proteins that play a key role in controlling the cell cycle. Abnormalities of cell cycle regulators, including cyclins and cyclin dependent kinases, have been reported in various malignant tumors. This study was undertaken to quantitatively detect cyclin B1 an...
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| Zitieren: | Expression profiling of cyclin B1 and D1 in cervical carcinoma / M. Zhao, Y.T. Kim, // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 44-48. — Бібліогр.: 23 назв. — англ. |
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irk-123456789-1341572018-06-13T03:03:35Z Expression profiling of cyclin B1 and D1 in cervical carcinoma Zhao, M. Kim, Y.T. Yoon, B.S. Kim, S.W. Kang, M.H. Kim, S.H. Kim, J.H. Kim, J.W. Park, Y.W. Original contributions Aim: Cyclins are a family of regulatory proteins that play a key role in controlling the cell cycle. Abnormalities of cell cycle regulators, including cyclins and cyclin dependent kinases, have been reported in various malignant tumors. This study was undertaken to quantitatively detect cyclin B1 and D1 in cervical cancer. Methods: A quantitative real-time reverse transcription polymerase chain reaction and Western blot assay were used to analyze the expression of cyclin B1/D1 mRNA and proteins, respectively, in fresh invasive cervical cancer (n = 41) and normal cervical tissues (n = 10). Results: There was significantly greater cyclin B1 expression in invasive cervical cancer than in normal cervical tissue (P = 0.019). However, cyclin D1 expression was not significantly different. A Western blot assay yielded similar results. Conclusion: Our results were consistent with the concept that up-regulation of cyclin B1 expression occurred in cervical cancer and an aberrant expression of cyclin B1 might play an important role in cervical carcinogenesis. Цель: циклины представляют собой семейство регуляторных белков, контролирующих клеточный цикл. Наличие функциональных и структурных нарушений регуляторов клеточного цикла (циклинов и циклинзависимых киназ) было отмечено в клетках различных злокачественных новообразований. Целью данного исследования было проведение количественного определения циклинов B1 и D1 в клетках рака шейки матки. Методы: определение уровня экспрессии циклинов B1/D1 (mRNA и белков соответственно) в свежеполученных клетках инвазивного рака шейки матки (n = 41) и нормальной ткани шейки матки (n = 10) проводили методами RT-PCR в режиме реального времени и Вестерн-блот анализа. Результаты: отмечен более высокий уровень экспрессии гена циклина В1 в клетках инвазивного рака шейки матки, чем в клетках нормальной ткани (P = 0,019). Не выявлены значительные различия в уровне экспрессии гена циклина D1. При Вестерн-блот анализе получены аналогичные результаты. Выводы: результаты исследования подтверждают концепцию об активации экспрессии циклина В1 при раке шейки матки. Аберрантная экспрессия циклина В1 может играть важную роль при злокачественной трансформации эпителия шейки матки. 2006 Article Expression profiling of cyclin B1 and D1 in cervical carcinoma / M. Zhao, Y.T. Kim, // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 44-48. — Бібліогр.: 23 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/134157 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Zhao, M. Kim, Y.T. Yoon, B.S. Kim, S.W. Kang, M.H. Kim, S.H. Kim, J.H. Kim, J.W. Park, Y.W. Expression profiling of cyclin B1 and D1 in cervical carcinoma Experimental Oncology |
| description |
Aim: Cyclins are a family of regulatory proteins that play a key role in controlling the cell cycle. Abnormalities of cell cycle regulators, including cyclins and cyclin dependent kinases, have been reported in various malignant tumors. This study was undertaken to quantitatively detect cyclin B1 and D1 in cervical cancer. Methods: A quantitative real-time reverse transcription polymerase chain reaction and Western blot assay were used to analyze the expression of cyclin B1/D1 mRNA and proteins, respectively, in fresh invasive cervical cancer (n = 41) and normal cervical tissues (n = 10). Results: There was significantly greater cyclin B1 expression in invasive cervical cancer than in normal cervical tissue (P = 0.019). However, cyclin D1 expression was not significantly different. A Western blot assay yielded similar results. Conclusion: Our results were consistent with the concept that up-regulation of cyclin B1 expression occurred in cervical cancer and an aberrant expression of cyclin B1 might play an important role in cervical carcinogenesis. |
| format |
Article |
| author |
Zhao, M. Kim, Y.T. Yoon, B.S. Kim, S.W. Kang, M.H. Kim, S.H. Kim, J.H. Kim, J.W. Park, Y.W. |
| author_facet |
Zhao, M. Kim, Y.T. Yoon, B.S. Kim, S.W. Kang, M.H. Kim, S.H. Kim, J.H. Kim, J.W. Park, Y.W. |
| author_sort |
Zhao, M. |
| title |
Expression profiling of cyclin B1 and D1 in cervical carcinoma |
| title_short |
Expression profiling of cyclin B1 and D1 in cervical carcinoma |
| title_full |
Expression profiling of cyclin B1 and D1 in cervical carcinoma |
| title_fullStr |
Expression profiling of cyclin B1 and D1 in cervical carcinoma |
| title_full_unstemmed |
Expression profiling of cyclin B1 and D1 in cervical carcinoma |
| title_sort |
expression profiling of cyclin b1 and d1 in cervical carcinoma |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2006 |
| topic_facet |
Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/134157 |
| citation_txt |
Expression profiling of cyclin B1 and D1 in cervical carcinoma / M. Zhao, Y.T. Kim, // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 44-48. — Бібліогр.: 23 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-09T20:25:18Z |
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2025-07-09T20:25:18Z |
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| fulltext |
44 Experimental Oncology 28, 44–48, 2006 (March)
Cervical cancer is an important public health
problem. It is the second most common malignancy
among women worldwide and the first in many
developing countries, including Korea [1]. It is widely
accepted that high-risk type human pappillomavirus
(HPV) infection is an obligatory factor in malignant
transformation within the epithelial cells of the uterine
cervix [2]. However, HPV infection alone is insufficient
to cause invasive cervical cancer [3]. The role of
promoting cofactors in the later stages of progression
into cervical malignancy remains uncertain. Possible
candidates are the cyclin proteins that are cyclically
synthesized and destroyed during the cell cycle.
There are many well-known cyclins, including 11
major classes and some subclasses, labeled A, B1-2,
C, D1-3, E1-2, F, G1-2, H, I, K, T1-2 to H. They share a
conserved amino acid region, known as the cyclin box,
through which they interact with their cyclin dependent
kinases (cdks) [4, 5]. Cyclins are also grouped accord-
ing to their phase of activity. The G1 cyclins include D1
and E, which are active in the G1 phase and regulate the
G1–S phase transition. Cyclin A is active in the S and
late G2 phase, while B1-2 are active in the G2 phase and
regulate mitosis. Cell cycle dysregulation is a feature of
all human cancers and there is evidence suggesting a
role for cyclins in human cancer [6].
The human B-type class of cyclins consists of three
closely related members, cyclins B1, B2, and B3, which
control the G2-M transition through interaction with the
cdk1 protein kinase.
Cyclin B1 was the first human cyclin identified and
is the best characterized among the three members
[7]. Cyclin B1 complexes solely with cdk1 (cdc2) to
form the mitosis-promoting factor, which regulates
the G2–M transition and is the primary regulator of
mitosis. The mitosis-promoting factor is located in
the cytoplasm late in the S phase, but relocates to the
nucleus shortly before the envelope breakdown. P53
was recently shown to prevent the G2–M transition by
decreasing cyclin B levels and attenuating activity of
the cyclin B promoter [5, 8, 9].
The cyclin D1 protein is a major positive regulator
of the progression from the G1 to S phase.There is a
homeostatic feedback loop between cyclin D1, Rb,
and p27Kip1 [5,10]. Cyclin D1 is often found disrupted
in the cancer cell genome by chromosome 11q13
translocation or gene amplification. Approximately
50% of mammary carcinomas have been shown to
overexpress the cyclin D1 protein. Molecular aberra-
tions accounting for increased mRNA transcripts of cy-
clin D1 in other cancers include gene rearrangements
and amplification. Amplification has been implicated
in many tumors, including bladder and ovarian cancer
[11]. In contrast, our previous study suggested that
there is no correlation between gene alterations and
protein D1 in cervical squamous cell cancer [12].
Previously, we studied a large collection of fresh or
paraffin-embedded samples from patients with cervical
cancer to analyze the expression levels of cyclin D1, E,
A by reverse transcription polymerase chain reaction
(RT-PCR) and immunohistochemical staining methods
[12–14]. We found that RT-PCR or immunohistochemical
assay could detect the cyclins message in nearly all the
cervical cancer specimens. However, these methods
lacked specificity and only yielded a semi-quantitative
result. Gene expression analysis requires sensitive,
precise, and reproducible measurement. Hence, we
reported the development of a quantitative real-time
RT-PCR assay for cyclin B1 and D1 expression with
high sensitivity and specificity for cervical cancer. Our
objective was to perform a quantitative real-time RT-PCR
assay in normal and cervical cancer tissues to evaluate
the expression level and status of cyclin B1 and D1.
ExprEssion profiling of CyClin B1 and d1 in CErviCal
CarCinoma
M. Zhao, Y.T. Kim*, B.S. Yoon, S.W. Kim, M.H. Kang, S.H. Kim, J.H. Kim, J. W. Kim, Y.W. Park
Department of Obstetrics and Gynecology, Women’s life and Science Institute,
Yonsei University College of Medicine, Seoul, Korea
Aim: Cyclins are a family of regulatory proteins that play a key role in controlling the cell cycle. Abnormalities of cell cycle regulators,
including cyclins and cyclin dependent kinases, have been reported in various malignant tumors. This study was undertaken to
quantitatively detect cyclin B1 and D1 in cervical cancer. Methods: A quantitative real-time reverse transcription polymerase chain
reaction and Western blot assay were used to analyze the expression of cyclin B1/D1 mRNA and proteins, respectively, in fresh invasive
cervical cancer (n = 41) and normal cervical tissues (n = 10). Results: There was significantly greater cyclin B1 expression in invasive
cervical cancer than in normal cervical tissue (P = 0.019). However, cyclin D1 expression was not significantly different. A Western
blot assay yielded similar results. Conclusion: Our results were consistent with the concept that up-regulation of cyclin B1 expression
occurred in cervical cancer and an aberrant expression of cyclin B1 might play an important role in cervical carcinogenesis.
Key Words: cyclin B1, cyclin D1, cervical cancer, real-time RT-PCR, Western blot.
Received: October 27, 2005.
*Correspondence: Fax: 82-2-313-8357
E-mail: ytkchoi@yumc.yonsei.ac.kr
Abbreviations used: HPV — human papillomavirus; FAM — 6-car-
boxyfluorescein; GAPDH — glyceraldehyde–3–phosphate-de-
hydrogenase; RT-PCR — reverse transcription polymerase chain
reaction; TAMRA — 6-carboxytetramethylrhodamine.
Exp Oncol 2006
28, 1, 44–48
Experimental Oncology 28, 44–48, 2006 (March) 45
matErials and mEthods
Patients. Fresh cervical tissue samples were
obtained from 51 patients treated at Yonsei Medical
Center. These included 41 invasive cervical cancers
and 10 normal cervix tissues that served as the con-
trol. Of the 41 patients with cervical cancer, 9 cases
were stage I, 27 cases were stage II, and 5 cases were
stage III. Dissected tissue samples were immediately
frozen and stored in liquid nitrogen. The patients’ ages
ranged from 29 to 80 years with a mean of 53.08 years.
Regarding distribution according to stage, patients
with stage II cervical carcinoma were the most numer-
ous. According to cell type, 37 contained squamous
cells and 4 were adenocarcinomas. The HPV infection
was detected in 41 cervical carcinoma tissues, and the
HPV types included types 4, 11, 14, 16, 18, 33, 35, 45,
58, 63, 69 and others.
RNA isolation and cDNA preparation. For
RNA preparation, samples were disrupted into small
pieces and RNA was isolated from the tumor and
control samples using SV total RNA isolation system
(Promega). A total of 1 µg RNA of each sample was
reverse transcribed using an oligo d(T) primer and
RNase H- MMLV reverse transcriptase, according to
the manufacturer’s protocol (Fermentas). The reverse
transcription reaction contained the following in a 50 µl
volume: 200 units MMLV enzyme, 1× MMLV buffer,
20 units Rnasin, 0.5 µg oligo d(T), 2.5 mM dNTP
mix, and DNase treated total RNA. The reaction was
incubated at 70 °C for 10 min. The control and ref-
erence RNA′s were reverse-transcribed alongside
patient RNA for each run. The cDNA was stored at
–20 °C and was used for each PCR reaction. The
quality of the cDNA was confirmed by amplification
of glyceraldehyde–3–phosphate-dehydrogenase
(GAPDH) and only samples with consistent and strong
amplifications were included in the final analyses.
Analyses of gene expression by real-time
quantitative RT-PCR. The quantitation of mRNA levels
was carried out using a real-time fluorescence detection
method. The cDNA was prepared as described above
and amplified by PCR in the ABI prism 7700 sequence
detector (PE Biosystems, USA). The reaction mixture
for cyclin B1/D1 contains the following in a final volume
of 50 µl: 25 µl Taqman mix, 2.5 µl cyclin B1 or D1 primer
mix, 1 µg cDNA, and added free-water to 50 µl. The
quantitative real-time PCR was performed as follows: an
initial cycle for 10 min at 95 °C, followed by 50 bi-phasic
cycles of 15 s at 95 °C, and 1 min at 60 °C.
The primer and probe sequences were published
previously (Table 1) [15, 16]. All primer and probe
combinations were positioned to span an exon-exon
junction. The probes were labeled at the 5′ end with a
FAM (6-carboxyfluorescein) probe and at the 3′ end
with TAMRA (6-carboxytetramethylrhodamine), which
served as a quencher. Initial template concentration
was calculated from the cycle number when the
amount of PCR product passed a threshold set in the
exponential phase of the PCR reaction.
Table 1. The sequences of primer and probe of cyclin B1 and D1
Cyclin B1 :
Forward primer: 5′—CTC CTG TCT GGT GGG AGGA—3′
Reverse primer: 5′—CTG ATC CAG AAT AAC ACC TGA—3′
Probe: 5′—FAM-AGA GTG GAG TTG TGC TGG CT-TAMRA—3′
Cyclin D1 :
Forward primer: 5′—CTG GCC ATG AAC TAC GTG GA—3′
Forward primer: 5′—GTC ACA CTT GAT CAC TCT GG—3′
Probe: 5′—FAM-AGA AGC GTG TGA GGC GGT AGT AGG A-TAMRA—3′
GAPDH :
Forward primer: 5′—GAA GGT GAA GGT GGG AGT C—3′
Forward primer: 5′—GAA GAT GGT GAT GGG ATT TC—3′
Probe: 5′—FAM-CAA GGT TCC CGT TCT CAG CC-TAMRA—3′
The threshold cycles (Ct) were recorded for
the target gene and reference in all the samples.
The results were calculated relative to a reference
standard, called a calibrator. The results of the real-
time PCR were calculated by “Comparative Ct method
of Quantitation” (ΔΔCt) [17, 18]. This method was
outlined in the ABI Prism Sequence Detection System
User Bulletin from the following formulas:
ΔCt (sample) = Ct (target gene) – Ct (GADPH),
ΔΔCt = ΔCt (tumor) – ΔCt (control)
Relative expression = 2-ΔΔCt
The three targets were analyzed separately. Then,
cyclin B1 and D1 were each normalized to GADPH as an
internal standard. Final relative cyclin B1/D1 expression
levels of each unknown sample were obtained by
division of cyclin B1/D1 copy numbers by GADPH copy
numbers. The normalization accounts for variability in
the samples’ reverse transcription efficiency and RNA
quantity and quality. The ΔΔCt was calculated separately
for cyclin B1 and cyclin D1, for each sample, using the
cell line HeLa as the calibrator. HeLa was chosen as the
reference calibrator because it expressed high levels of
cyclin B1 or cyclin D1, and it is potentially a consistent
source of calibrator template. At least two independent
analyses were performed for each sample and each
gene. Analyses of the gene expression data were
performed without knowledge of the patients′ data.
Real-time PCR efficiency assay. To use the
2-ΔΔCt calculation, the PCR efficiencies of the target
and control assays need to be similar [17]. The
relative efficiencies were measured by varying the
input amount of RNA, measuring the Ct values, and
calculating the ΔCt values for the various assays.
Graphing the ΔCt values relative to input total RNA
should give a line with a slope < 0.1. The range of input
RNA should be from 10 ng to 1000 ng.
Western blot analysis of cyclin B1 and D1. The
cervical tissues were all homogenized and then lysed
with cell lysis buffer (Cell Signaling Technology, Inc),
according to the manufacturer protocol. After incuba-
tion on ice for 10 min, tissue lysates were clarified by
centrifugation at 13,000 rpm for five min and protein
concentration of the supernatants was determined
with a Bio-Rad DC protein assay (Bio-Rad, USA).
40 µg of total protein was loaded in each lane. The
samples were subjected to 8–10% SDS-PAGE. The
resolved proteins were blotted to a PVDF membrane,
46 Experimental Oncology 28, 44–48, 2006 (March)
which were then blocked at overnight 4 °C in TBS buf-
fer (20 mM Tris base, 137 mM sodium chloride, 1 M
hydrochloric acid, pH 7.6) containing 5% non fat milk.
The membrane was then incubated with the primary
antibodies: mouse-anti cyclinB1 (clone V152, Cell
Signaling Technology, Inc), anti-human cyclinD1
(clone Dcs6, Cell Signaling Technology, Inc) and
mouse-anti GADPH (Cell Signaling Technology, Inc)
for 1 h at room temperature. The concentration of
each antibody was used as suggested by the suppli-
ers. Peroxidase labeled anti-mouse antibodies were
used as a secondary antibody, diluted in TBS with 5%
(w/v) milk, and developed using the chemiluminescent
ECL kit and Hyperfilm ECL (Amersham Biosciences,
USA). Quantification of proteins was performed by
using a laser densitometer and analysis software (IM-
AGE READER LAS-1000 lite, Fuji Photo Film Co., Ltd.,
Japan), while referring to the standard series. The den-
sitometric integral derived from each sample band, i.e.
the integral of a mean density over a measured area,
was taken to calculate the amount in each sample,
according to the known standard values.
Statistical analyses. Data were analyzed using
parametric and nonparametric statistics, SPSS 11.0
(Chicago, USA). Descriptive statistics were used for
quantitative experimental data and are summarized as
means and standard deviations. Continuous variables
were examined for a normal distribution (Kolmogorov-
Smirnov test) before adopting parametric statistics.
Differences between continuous variables were
evaluated by an independent-sample T test. One way
ANOVA and logistic regression analysis were done for
normally distributed variables. The Mann-Whitney U
test and the Kruskal-Wallis test were done for variables
that were not normally distributed. Differences were
considered significant when the probability of error
was below 5% (P < 0.05).
rEsults
Analysis of cyclin expression in normal and
cervical cancer tissues with real-time RT-PCR.
To determine cyclin B1 and D1 transcript level in fresh
human cervical carcinoma tissues, we used a real-time
quantitative RT-PCR assay based on TaqMan method-
ology. GAPDH mRNA was measured, as a reference,
to normalize cyclin B1 and D1 mRNA levels. Primers
and probes were selected to avoid amplification from
genomic DNA and target sequences were kept small
to ensure the detection of fragmented and partially
degraded RNA. The PCR efficiencies of the target
and control assays were performed. The slope for
cyclin B1 was 0.088 and the slope for cyclin D1 was
0.094, relative to GAPDH for the range of input cDNA
of 10–1000 ng.
In the study, we compared cyclin B1 and D1 expres-
sion in cervical cancer and normal tissues. These anal-
yses revealed that cyclin B1 transcripts had a higher
expression in cervical cancer (P = 0.019) and there was
a decreased trend from stage I (0.76 ± 0.68) to stage
III (0.23 ± 0.39) in cervical cancer cases (Table 2).
Although we found a high level of cyclin D1 expression
in some cervical cancer cases, no significant statistical
differences were observed. In addition, we also found
an increase in cyclin D1 mRNA expression from stage I
(2.17 ± 2.33) to III (6.66 ± 10.48) in cervical cancer
cases. Moreover, no significant differences in terms
of cyclin expression were found between the different
stages of cervical carcinoma.
Table 2. Expression of cyclin B1 and D1 in the uterine cervix
Tissue Cyclin B1 Cyclin D1
2– ∆ ∆ Ct value (mean ± SD) 2– ∆ ∆ Ct value (mean ± SD)
Control (n = 10)
Cervical cancer (n = 41)
Stage I (n = 9)
Stage II (n = 27)
Stage III (n = 5)
0.03 ± 0.05
0.59 ± 0.72a
0.76 ± 0.68
0.57 ± 0.76
0.23 ± 0.39
3.22 ± 2.29
3.29 ± 4.63
2.17 ± 2.33
3.24 ± 4.32
6.66 ± 10.48
aP = 0.019 (cervical cancer vs. control), Students t-test.
Western blotting of cyclin B1 and D1 in cervical
carcinoma. The mRNA expression level of cyclin B1
and D1 were analyzed with real-time RT-PCR. We
wanted to directly detect cyclin B1 and D1 protein
expression. So, we selected 29 cases of cervical
cancer and 6 cases of normal cervical tissues in which
mRNA expression was analyzed by real-time RT-PCR.
In western blotting, cyclin B1 protein was detected
as a 60 KDa and cyclin D1 protein was 36 KDa band
(Figure). Then, we compared the expression levels of
mRNA with protein expression levels of cyclinB1 and
D1 in cervix tissue with and without cervical cancer.
Our study revealed a statistically significant increase
in expression of cyclin B1 in patient with cervical
compared to normal cervix tissue (P = 0.031) (Table 3).
These results agreed with the results of the mRNA
analysis by real-time PCR. There was no statistical
difference in expression of cyclin D1 between cervical
cancer and normal tissues, and no correlation between
the expression of mRNA and protein.
Table 3. The protein analysis of cyclin B1and D1 by Western blot
Tissue Cyclin B1 (mean ± SD) Cyclin D1 (mean ± SD)
Control
Cervical cancer
0.21 ± 0.11
0.33 ± 0.11a
0.27 ± 0.16
0.32 ± 0.20
aP = 0.031 (cancer vs. control), Students t-test.
disCussion
Exact quantification of cyclin B1/D1 expression
levels might elucidate the causes and consequences
of cyclin B1 or D1 deregulation in cervical cancer. The
quantitative real-time RT-PCR assay described in this
report represents a major improvement over other
assays designed to quantify cyclin B1/D1 expression
in tissue specimens. This assay can detect the PCR
product following each cycle of the reaction, during
the linear range of amplification, then eliminates the
need for post PCR analysis. Real-time RT-PCR assay
is an ideal tool for the detection and quantification of
cyclins in cervical carcinoma because it is reliable,
rapid, sensitive, and specific. Another advantage is its
applicability to the degraded nucleic acids obtained
from fixed and embedded cervical tissue specimens,
which neither Southern nor Northern blotting of tissue
RNA can be performed successfully [17, 18]. In brief,
real-time RT-PCR combined the speed and ease of a
Experimental Oncology 28, 44–48, 2006 (March) 47
PCR-based system with an accurate and reproducible
quantification methodology and therefore has the po-
tential to become a routine diagnostic tool.
figure. Western blot of cyclin B1 and D1 (a) and boxplot graphs
demonstrating the cyclin B1 and cyclin D1 protein expression in
normal cervical and cervical carcinoma tissues. (a): Western blot
of cyclin B1 and D1 in normal and cancer cervical tissues. Protein
extracts were separated by 10% SDS–PAGE, transferred from
the gel to PVDF membranes, and immunoblotted with cyclin B1
and D1 monoclonal antibodies (HeLa: HeLa cell line; C: control
tissue; T1-4: tumor samples). (b): Boxplot graphs demonstrating
cyclin B1 and cyclin D1 protein expression in normal cervix and
cervical carcinoma (Western blot). The lines across the boxes
represent the medians and the whiskers extend to the highest and
lowest values, excluding outliers. The small circles and asterisks
identify outliers and extreme values.
In this study, results were calculated by the by com-
parative Ct method of quantification (2-ΔΔCt method).
The relative expression of cyclin and GAPDH genes
could be estimated from the difference in Ct values
(ΔCt). The ΔCt value would be normalized to a control
by subtracting the ΔCt value obtained with the calibrator
to yield a ΔΔCt. Ct values for cyclin B1/D1 and GAPDH
in the test tissues were compared with those obtained
with the HeLa cell line. The greater the ΔΔCt value, the
lower the expression of cyclin B1/D1 in the specimens.
The 2-ΔΔCt method is a convenient method to analyze
the relative changes in gene expression from real-time
quantitative RT-PCR experiments.
The main purpose of this study was to evaluate the
potential link between the expression of cyclin B1/D1
and cervical tumorigenesis. As a key cell cycle modu-
lator of the G2-M transition, cyclin B1 is considered
to play an important role in various human tumors.
However, to the best of our knowledge, this is the first
report to demonstrate the up-regulation of cyclin B1
expression in patients with cervical carcinoma using
quantitative real-time RT-PCR assay. This finding was
in agreement with the previously published study by
Kanai et al. They reported elevated cyclin B1 levels in
invasive carcinomas and concomitantly elevated levels
of cdk1 in the immunohistochemical study [19].
On the other hand, no significant difference in the
expression of cyclin B1 was found among various
clinicopathological parameters including histology,
histological subtype, tumor lesion, and patient’s age.
Furthermore, we found that there was a trend of decrease
in the mRNA expression of cyclin B1 with each advancing
stage. Other studies have also shown that in high-grade
cervical precancerous lesions, cyclin B1 expression was
up-regulated and persists into the upper epithelial layers
[4]. This suggests that cyclin B1 could play a crucial role
in the early phase of cervical carcinogenesis.
Because it is not clearly understood, there are
controversial results on the role of cyclin D1 in cervi-
cal carcinogenesis and clinical outcome [20, 21]. The
hypophosphorylated form of Rb, complexed with E2F,
serves as activator of cyclin D1 transcription by binding
to its promoter. This drives cyclin D1 in the early and mid
G1 phase of the cell cycle. Since D type cyclins and HPV
E7 possess similar binding regions for pRb and pRb-
related pocket proteins, inactivation of pRb either by the
cyclin/cdk complexes in G1 or by interaction with the
high-risk HPV oncoprotein E7 may result in a decreased
expression of cyclin D1 [6, 22]. In this study, we found
no significant difference in cyclin D1 mRNA expression
between cervical cancer and normal cervical tissues.
This finding was slightly different from previous stud-
ies, which described significantly lower in HPV-positive
cervical lesions compared to HPV-negative cases and
normal cervical epithelium [22]. Different detection
methods and analysis standards may have caused the
conflicting observations because semi-quantitative as-
says, including immunohistochemical assay, RT-PCR,
and Western analysis, were used to detect expression
levels of cyclins and other molecules [23]. Further study
of this phenomenon is warranted.
In summary, this study demonstrated that cyclin
B1 mRNA expression was significantly increased in
invasive cervical carcinomas compared with normal
cervix. This change may play a role in uncontrolled
proliferation and malignant transformation of the
uterine cervix. However, additional molecular studies
to identify the amplification of the cyclin B1 and D1
48 Experimental Oncology 28, 44–48, 2006 (March)
genes or the source of cyclin protein overaccumulation
will be necessary to clarify these findings.
aCknowlEdgmEnts
This study was supported by Brain Korea (BK) 21
Project for Medical Sciences Yonsei University and a
grant of the Korea Health 21 R& D Project, Ministry
of Health & Welfare, Republic of Korea (0412-CR01-
0704-0001).
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особенности экспрессии циклинов B1 и d1
при рАке шейки мАтки
Цель: циклины представляют собой семейство регуляторных белков, контролирующих клеточный цикл. Наличие
функциональных и структурных нарушений регуляторов клеточного цикла (циклинов и циклинзависимых киназ) было
отмечено в клетках различных злокачественных новообразований. Целью данного исследования было проведение
количественного определения циклинов B1 и D1 в клетках рака шейки матки. Методы: определение уровня экспрессии
циклинов B1/D1 (mRNA и белков соответственно) в свежеполученных клетках инвазивного рака шейки матки (n = 41) и
нормальной ткани шейки матки (n = 10) проводили методами RT-PCR в режиме реального времени и Вестерн-блот анализа.
Результаты: отмечен более высокий уровень экспрессии гена циклина В1 в клетках инвазивного рака шейки матки, чем в
клетках нормальной ткани (P = 0,019). Не выявлены значительные различия в уровне экспрессии гена циклина D1. При
Вестерн-блот анализе получены аналогичные результаты. Выводы: результаты исследования подтверждают концепцию
об активации экспрессии циклина В1 при раке шейки матки. Аберрантная экспрессия циклина В1 может играть важную
роль при злокачественной трансформации эпителия шейки матки.
Ключевые слова: циклин В1, циклин D1, рак шейки матки, RT-PCR в режиме реального времени, Вестерн- блоттинг.
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