Download Differential expression of cyclin D1 in human cancer cell and normal

Differential expression of cyclin D1 in human cancer cells and normal fibroblast
cells
Jing Yang
Department of Biology
Fordham University
Bronx, NY
Abstract:
Cyclin D1 is the first cyclin expressed in G1 phase of the cell cycle in response to
mitogens. It binds to Cdk4 or Cdk6 and the holoenzyme phosphorylates Rb to induce the
cell cycle progression from G1 to S phase. Cyclin D1 is rate limiting for G1 phase
progression in many cell types and is over-expressed in various human cancer cells,
which underscores its role in the cell transformation. In this study, I compared the mRNA
expression levels of cyclin D1 using RT-PCR in a human cancer cell line (HeLa) and in a
humanl fibroblast cell line (WI-38). My results indicate that there is no significant
difference in cyclin D1 mRNA expression in these two cell lines, suggesting that cyclin
D1 over-expression may not be necessary for the tumorigenesis of human cervical
cancers and may not be a good target for cervical cancer therapy.
Introduction:
Cyclin D1 is known as a critical regulatory protein in the cell cycle. It belongs to
the D type cyclin family that also includes cyclin D2 and cyclin D3. The expression of
cyclin D1 is ubiquitous whereas cyclin D2 and D3 expression are more restricted. The
protein level of cyclin D1 oscillates periodically during the cell cycle with the highest in
G1 phase and lowest in late S phase. In eukaryotic cells, the mRNA and protein levels of
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cyclin D1 are increased when the cell is stimulated by mitogen stimulations. Once it is
induced, cyclin D1 interacts with Cdk4 and Cdk6 in early G1 phase to activate the kinase
activity of Cdk4 and Cdk6 (1), which is critical for G1 to S phase cell cycle progression.
The cyclin D1/Cdk4,6 complex phosphorylates various targets but the primary target is
believed to be the retinoblastoma protein (Rb), one of the key inhibitors of cell cycle
progression, to promote cell cycle progression (1). Hyperphosphorylated Rb protein loses
its ability to interact with E2F family transcriptional factors whose target genes are
critical for cell cycle progression. Thus, the cyclin D1-Rb-E2F pathway is critical for the
cell cycle progression from G1 to S phase. Immunoneutralization and anti-sense
experiments have demonstrated that the abundance of cyclin D1 is rate limiting for G1
progression in many cell types, including fibroblasts and human breast epithelial cells.
Cyclin D1 is dispensable for G1 progression in cultured mammalian cells that lack
functional Rb protein, again underscoring that Rb is the major target of cyclin D1 activity
(2).
The kinase activity of cyclin D1/Cdk4,6 complex is controlled by p16, one of the
members of the ink4a cyclin dependent kinase inhibitor (CKI) family. Upon the
interaction of p16 with Cdk4 or Cdk6, p16 is displaced from the complex so that the
kinase activity of the complex is diminished. It was also reported that cyclin D1/Cdk4,6
complex can bind to the kip1 CKI family protein p21 and p27 to modulate their activities.
In addition, recent studies indicated that cyclin D1 possesses functions independent of
Cdk4 and Cdk6. In this capacity, cyclin D1 is discovered to serve as a transcriptional
factor to activate the transcription of a variety of transcriptional factors (3).
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Alteration of cyclin D1 gene expression is frequently observed in human cancers.
In fact, most of the human cancers have defects in cyclin D1-Rb-E2F pathway. Genetic
and immunohistochemical studies have demonstrated that cyclin D1 is over-expressed in
human breast cancer (4,5), esophageal cancer (6) and human thyroid cancer(7). To
further investigate the differential expression pattern of cyclin D1 in human cancer cells,
I compared the gene expression levels of cyclin D1 in HeLa and WI-38 cells, a human
cervical cancer cell line and a human fibroblast cell line, respectively. Preliminary results
indicated that the mRNA level of cyclin D1 is not upregulated in HeLa cells as compared
to WI-38 cells. Although further experiments need to be performed, I conclude that
cyclin D1 may not be essential for the oncogenesis of human cervical cancers.
Materials and Methods:
Cell lines
HeLa and WI-38 cells were provided by Jingsong Qiu of Dr. Berish Rubin’s lab.
Both cell lines were grown in DMEM supplemented with 10% fetal bovine serum at 370C
in a humidified atmosphere with 5% CO2.
Primers
Primers specific for cyclin D1 were purchased from Invitrogen. The forward
primer (cd-f1) is located in exon 4 with the sequence 5’CAAAATGCCAGAGGCGGAG-3’ which corresponds to nucleotides 707-728 of
accession number NM_053056. The reverse primer (cd-r1) is located in exon 5 with the
sequence 5’-CTTGATCACTCTGGAGAG-3’ which corresponds to nucleotides 906-923
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of accession number NM_053056. The primers were expected to generate a 217 base pair
product.
RNA extraction:
RNA samples from HeLa and WI-38 cells were provided by Jingsong Qiu of Dr.
Berish Rubin’s lab.
RT-PCR
RT-PCR was performed on the RNAs prepared from HeLa and WI-38 cells using
the Qiagen One-Step RT-PCR Kit. Each reaction contains 3µl 5X RT buffer, 0.6µl
10mM dNTPs, 0.75µl primer (10pmol/µl), 0.6µl enzyme mix, 5µl RNA template (with a
concentration of 20ng/µl) and 6.3 µl dH2O to a final volume of 15µl. RT-PCR was
performed based on the following program: 50ºC x 30 min and 95ºC x 15 min for one
cycle, followed by amplification at 94ºC x 20 sec, 59ºC x 30 sec and 72ºC x 30 sec for 34
cycles for cyclin D1 and 27 cycles for GAPDH. PCR products were analyzed on a 1%
agarose gel run at 130 volts for 45 min.
PCR purification
PCR products were purified using the Qiagen Rapid PCR Purification System
according to the manufacturer’s instructions.
Sequencing
The Sanger dideoxy method was used to sequence the purified PCR products with
AmpliCycle Sequencing kit (Applied Biosystems). Each reaction was done as follows: 50
fmol of PCR product was mixed with 4µl of 10X cycling buffer, 2µl of primer, 0.2µl of
α-33P-ATP and dH2O to a final volume of 30µl. 6µl of the master mix was added to four
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tubes containing 2µl of either ddGTP, ddATP, ddTTP or dCTP. All of the reaction
mixtures were overlaid with 12µl of mineral oil to prevent evaporation. These reactions
were set up using both the forward and reverse cyclin D1 primers. After mixing of the
sequencing components, the following program was performed: 94ºC x 3 min, followed
by 35 cycles of 94ºC x 30 sec, 58ºC x 30 sec and 72ºC x 1 min. 4µl of Stop Solution was
added to each tube and the samples were denatured by heating for 3 min after sequencing
program. The products were electrophoresed on a denaturing polyacrylamide gel and the
nucleotide sequence was visualized by autoradiography. The sequence obtained was
aligned with the cyclin D1 mRNA sequence from NCBI GenBank (NM_053056).
Results:
mRNA expression levels of cyclin D1 in HeLa and WI38 cells
To compare the mRNA expression pattern of cyclin D1 in cancer cells versus
normal ones. I performed RT-PCR detecting cyclin D1 in a human cancer cell line, HeLa
,and in a human fibroblast cell line, WI-38. The primers for the RT-PCR experiment were
designed to span regions between exon 4 and exon 5. In this experiment, GAPDH was
used as a control. The PCR products were loaded on a 1% agarose gel. As shown in
Figure 1, the mRNA levels of cyclin D1 show no significant difference between HeLa
and WI-38 cells. GAPDH mRNA level showed that the equal amounts of RNA were used
in the PCR reaction(left panel).
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WI-38
Hela
WI-38
Hela
Cyclin D1
GAPDH
Figure 1. RT-PCR analysis of cyclin D1 mRNA level in HeLa and WI-38 cells. The
expected size of cyclin D1 was 217 base pairs. PCR products were analyzed on a 1%
agarose gel run at 130 volts for 45 min. GAPDH was used as a loading control.
Confirmation of PCR product by sequencing
The PCR products were purified and sequenced by Sanger Dideoxy method to
confirm the identity. The 217 base pair PCR product was then aligned with Homo
Sapiens mRNA sequence of cyclin D1 (NM_053056) from NCBI. Figure 2 showed that
one hundred percent homology between 217 base pairs RT-PCR product and cyclin D1
mRNA (NM_053056), which demonstrated that the product is amplified from the
expected region of cyclin D1 mRNA.
RT-PCR product:
Cyclin D1 mRNA:
RT-PCR product:
Cyclin D1 mRNA:
RT-PCR product:
Cyclin D1 mRNA:
RT-PCR product:
Cyclin D1 mRNA:
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Figure 2. Alignment of RT-PCR product with cyclin D1 genebank NM_053056. One
hundred percent of homology was identified between these two sequences.
Discussion:
In this study, I compared the mRNA expression level of a human cancer cell line
HeLa and a human normal fibroblast cell line WI-38 using RT-PCR technique. Previous
studies have shown that cyclin D1 over-expression is a marker for many cancer cells (8).
Forced expression of cyclin D1 in Rat6 cell, which is a normal rat fibroblast cell, can lead
to the transformation of Rat6 cells (9). All of these information underscore the
significance of cyclin D1 in promoting of the cell proliferation and oncogenesis. In this
study, however, the cyclin D1 mRNA level was not higher in HeLa cells than that of WI38 cells.
It is reported that cyclin D1 level is controlled both in the mRNA level and in the
protein level (10). Therefore, western blot analysis is required to determine whether the
protein level of cyclin D1 is increased in HeLa cells compared with WI-38 cells. The
level of cyclin D1 is also controlled in the cell cycle. Therefore, the cell cycle profile of
HeLa and WI-38 cells before harvest could also affect the result.
The cyclin D1 protein and coding sequence from the majority of tumors examined
are normal without mutations, which suggests that the over-expression of wild-type
cyclin D1 is responsible for the formation of tumors (11). The functional inactivation of
Rb through deregulated phosphorylation may thus be potentiated in a broad variety of
tumors by the over-expression of wild-type cyclin D1 (12). My result is consistent with
this idea by showing that the amplified sequence from cyclin D1 mRNA harbors no
mutations when aligned with cyclin D1 mRNA from NCBI genebank.
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Although additional experiments are required to further confirm this finding, my
results indicate that cyclin D1 may not a critical oncoprotein in the oncogenesis of human
cervical cancer (HeLa cell). This is also consistent with the idea that any single mutation
in cyclin D1-Rb-E2F pathway can lead to oncogenesis. In this respect, it is known that
HeLa cells have altered function of Rb protein (13) and the activity of E2F in HeLa cells
is up-regulated (14). Indeed, detailed analysis of cyclin D1 expression profiles and cyclin
D1-Rb-E2F pathway in different cancer cells will reveal how deregulation of cyclin D1
will affect the proliferation and differentiation of cells.
Acknowledgement:
I would like to thank Jinsong and Lisa for their patience, guidance, and
encouragement during this project. Many thanks to Dr. Berish Rubin for providing the
cell lines. I also want to thank Dan for so generously help. Thanks to everyone who
gave me help and thanks Fordham University Department of Biology for funding this
research.
Reference:
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