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 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).
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.
Figure 2-Alignment of RT-PCR product with cyclin D1 Genbank NM_053056. One hundred percent of homology was identified between these two sequences.
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).
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.
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 WI-38 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 Genbank.
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.
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.
|This document was last modified 05/16/2006.|
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