The Effect Of Cyclin D1a/b On The Proliferation And Metastatic Potential Of Tumor Cell

Cyclin D1 shortens G1/S and contributes to human cancer. Alternately spliced forms from CCND1 mRNA were associated with increase cancer risk and poor prognosis in many cancers. By contrast, a recent study also found that the polymorphism had not a major gene effect on the given cancer and not associated with the pathogenesis of human cancers. The underlying mechanism of how cyclin D1 influenced the cancer prognosis is unclear. Besides, various factors and many-way mechanism often involve cancer risk and poor prognosis in many cancers. The proliferation of tumor cell is linked with the apoptosis of tumor cell. The apoptosis of tumor cell is a cellular self-destruction mechanism involved in a variety of biological events. The Bcl-2 family is a major regulator of mitochondria-regulated apoptotic events and plays a pivotal role in determining the eventual outcome. The Bcl-2 family is classified into the two subfamilies depending on the homology and functions of each and protein. One subfamily included Bcl-2, Bcl-xL, all of which exert anti-cell death activity prevented cytochrome C release by preserving mitochondrial membrane integrity and preventing pore formation and share sequence homology particularly within four regions. Another subfamily represented by Bax and Bid, all these proteins determine to a large extent that the cell initiates apoptosis induced cytochrome C release. The Bcl-2/Bax ratio constitutes a life or death decision point for cells. Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the proteolytic processing of extra cellular matrix structural proteins, which regulate cell migration. Matrix metalloproteinase-2/9 (MMP-2/9) is involved in the processes of tumor invasion and metastasis, and their overexpression is associated with poor prognosis. Integrins modulated cellular proliferation, apoptosis, migration, and invasiveness, especiallyαvβ3 integrin was associated with the aggressiveness and the prognosis of a variety of cancer cells, activated proMMP-2 and proMMP-9. Theαvβ3 integrin, functionally coupled with syndecan-1, is similar to the interaction of the syndecan-1 ectodomain with that promotes cell spreading. Theα5β1 integrin regulate upαvβ3 integrin in the help of syndecan-4. Cdc2 was a down stream effector of theαvβ3 integrin regulated up both expression of cdc2 mRNA and protein, and cdc2 associated with cyclin B2 regulates cell migration. Hox genes encode DNA-binding proteins which function as transcriptional regulators. The Hox D3 gene mediated up-regulation expression ofαv,α4,aIIb andβ3 integrin, bond fibronectin (FN)and vitronectin (VN), and influenced tumor invasion and metastasis. We have cloned cell lines expressing cyclin D1a and cyclin D1b, and explored how they respectively influence in human cancer.Method: 1. MCF-7 breast carcinoma cell lines of stable transfectants expressing cyclin D1a, cyclin D1b and pcDNA3.1, and BCL-7402 hepatocarcinoma cell lines of stable transfectants expressing cyclin D1a, cyclin D1b and pcDNA3.1, were measured respectively using MTT assay. Absorbance was determined at an OD of 570nm. Cells were stained respectively with CSFE. Parameters were acquired on a FACS Calibur flow cytometer and analyzed with CellQuest software after 72 h.2. Cells were induced respectively by 25μg/ml mitomycin c for 24 h, collected and washed softly twice with PBS and incubated with Annexin/PI for 30 min at 4℃. The cells were then washed twice with PBS. Parameters were acquired on a FACSCalibur flow cytometer and analyzed with CellQuest software. Total RNAs were isolated using TRIzol reagent from MCF-7 cells and BCL-7402 cells after treatment with 25μg/ml mitomycin c for 24 h. Equal amounts of Bcl-2, Bcl-xL, Bax and Bid mRNAs were reverse-transcribed and amplified by the PCR with specific primers. GAPDH mRNA was used as an internal control and co-amplified. Amplified products were analyzed by electrophoresis on 1.5% agarose gels and stained with ethidium bromide. 3. Nude mice were inoculated respectively by the right breast injection of 1×106 MCF-7 breast carcinoma cells and by the intra-liver injection of 3×105 BCL-7402 hepatocarcinoma cells. On d20, d30, d45 and d60 after inoculation of tumor cells in the right breast, the size of nodes was measured using calipers fitted with a vernier scale, and calculated using the formula (a+b)/2, with a as the larger diameter and b as the smaller diameter. On d30 and d60 after inoculation of tumor cells, the weight of nodes was measured in the right breast and in the intra-liver.4. MCF-7 breast carcinoma cell line of stable transfectants expressing cyclin D1a, cyclin D1b and pcDNA3.1, and BCL-7402 hepatocarcinoma cell line of stable transfectants expressing cyclin D1a, cyclin D1b and pcDNA3.1 was scratch respectively one separate wound, 3 mm in diemeter, and took pictures on following d1, d2 and d3. MCF-7 cells and BCL-7402 cells, 1×105 cells to each well in triplicate, were plated respectively on 96-well plates, which it was pre-coated with 10μg/ml of fibrinogen at different concentrations. The plates were incubated at 37℃for 2 h, and then washed three times with PBS to remove non-adhering cells. The relative amount of the adherent cells was measured by LDH assay. MCF-7 cells and BCL-7402 cells were put in 24-well fitted wells with membranes coated with ECM(5μg/cm2). Transwell migration assays were carried out in 5% CO2 at 37℃. After 48 h of incubation, cells from the lower surface were counted. 5. Nude mice were inoculated respectively by the right breast injection of 1×106 MCF-7 cells and by the intra-liver injection of 3×105 BCL-7402 cells. Lung, liver, blood, lymph, kidney, stomach, skin and bone from nude mice were measured the expression of cyclin D1a/b mRNAs by the RT- PCR. On d30 and d60 after inoculation of tumor cells, tumorous nodes formed in lung, liver, blood, lymph, kidney, stomach, skin and bone from nude mice were measured by HE staining and immunohistochemistry.6. Proteins of tumor cells were extracted from each group without or with stimulation of ECM. After the separation of proteins by 7.5% SDS-PAGE containing 1% gelatin, gels were incubated in MMP activation buffer overnight at 37℃and stained for 3 h and destained. The relative mRNA levels ofαv,β3, cdc2 and syndecan-1/4 were detected with real-time PCR amplified with SYBR Green Universal PCR Mastermix. MCF-7 cells and BCL-7402 cells were collected respectively and washed twice with PBS and incubated withαvβ3 antibody for 30 min at 4℃, and incubated with FITC-labeled anti-αvβ3 antibody for 30 min at 4℃. The cells were then washed twice with PBS and analyzed on a FACS Calibur flow cytometer. MCF-7 cells and BCL-7402 cells without or with stimulation of ECM were separated by SDS-PAGE followed by transfer onto nitrocellulose membranes. The membranes were blocked in TBST containing 5% nonfat milk, and probed with cdc2 and syndecan-1/4 polyclonal antibody. After incubation with the secondary antibody conjugated with horseradish peroxidase, membranes were detected using the ECL Western Blot Detection System. Proteins of MCF-7 cells were immunoprecipitated with anti-cyclin D1 antibody and immunoblotted with anti-HoxD3 antibody.Results: 1. MTT assay indicated that cyclin D1a groups exhibited a significantly increased ability to faster grow relative to cyclin D1b groups and pcDNA3.1 groups in MCF-7 cells and BCL-7402 cells. CSFE staining assay showed that cyclin D1a groups exhibited an increasing ability to faster grow relative to cyclin D1b groups and pcDNA3.1 groups in MCF-7 cells and BCL-7402 cells. In addition, cyclin D1b makes some cells to faster grow in MCF-7 cells and BCL-7402 cells.2. It indicated that rate of apoptosis of MCF-7 cells, pcDNA3.1 group 19.92% , cyclin D1a group 17.18%, and cyclin D1b group 15.84%. For BCL-7402 cells, pcDNA3.1 group 34.77%,cyclin D1a group 29.55%,cyclin D1b group 22.52%. There were no statistically differences in MCF-7 cells, but cyclin D1b group is resistent to apoptotsis of tumor cell in BCL-7402 cells. The expression of the Bcl-xL, Bax and Bid mRNAs in cell lines expressing amplified pcDNA3.1, cyclin D1a and cyclin D1b by PCR had no up or down regulation. However, it has the change between cyclin D1b and other groups for the Bcl-2 mRNAs of BCL-7402 cells.3. On d30 and d60, weight of tumor cells of cyclin D1a group were statistically different with cyclin D1b group and pcDNA3.1 group respectively among MCF-7 cells and among BCL-7402 cells. 4. Adhesion assay showed that the ability of cells expressing cyclin D1b binding to fibrinogen was significant compared with another cells. Scracth assay suggested that cells expressing cyclin D1b have stronger ability of migration than other cells. Transwell migration assays further confirm the phenomenon mensioned above.5. On d30 and d60, nude mice inoculated respectively MCF-7 cells and BCL-7402 cells were measured cyclin D1b mRNAs in lung and lymph, and On d60 nude mice inoculated MCF-7 cells formed minimal residual disease in lung, and On d90 nude mice inoculated MCF-7 cells formed tumorous nodes in lung.6. By gelatin zymography, both the production and the activity of MMP-2 and MMP-9 in tumor microenvironment were not different respectively in MCF-7 cells and in BCL-7402 cells without stimulation of ECM. However, with stimulation of ECM, both the production and the activity of proMMP-2/9 and active MMP-2/9 of pcDNA3.1 group of MCF-7 cells were not stronger than cyclin D1a group and cyclin D1b group, and active MMP-9 protein of cyclin D1a group was not stronger than cyclin D1b group, and proMMP-2 protein of cyclin D1a group was stronger than cyclin D1b group. Both the production and the activity of MMP-2 and MMP-9 of cyclin D1a group of BCL-7402 cells were not stronger than pcDNA3.1 group and cyclin D1b group, and proMMP-2 protein, active MMP-2 protein, proMMP-9 protein and active MMP-9 protein of cyclin D1b group were stronger than pcDNA3.1 group. The relative mRNA expression ofαv was up-regulation in cyclin D1a group of MCF-7 cells and BCL-7402 cells, the relative mRNA expression ofβ3 was up-regulation in cyclin D1b group of MCF-7 cells and BCL-7402 cells, and the relative protein expression ofαvβ3 was up-regulation in cyclin D1b group of MCF-7 cells and BCL-7402 cells. The relative mRNA expression of cdc2 without stimulation of ECM was up-regulation in cyclin D1a group of MCF-7 cells and BCL-7402 cells, the relative mRNA expression of cdc2 with stimulation of ECM was up-regulation in cyclin D1b group of MCF-7 cells and BCL-7402 cells, and it was the same in protein expression level. The relative mRNA expression of syndecan-1 was up-regulation in cyclin D1b group of MCF-7 cells and BCL-7402 cells, the relative mRNA expression of syndecan-4 had no difference among MCF-7 cells and BCL-7402 cells, and it was comfirmed by western blot. The production of proteins of cyclinD1b group of MCF-7 cells immunoprecipitated with anti-cyclin D1 antibody and immunoblotted with anti-HoxD3 antibody was more increased than other groups.Conclusion: Cyclin D1a had the marked effect on the cell proliferation in vitro and in vivo. It is not significant that cyclin D1b results in the cell proliferation and cyclin D1b is resistant to mitomycin c-induced apoptosis. Hox D3 binds cyclinD1 and mediated up-regulation expression of relative genes. Increasedαvβ3 integrin functionally coupled with syndecan-1 increase adhesion of tumor cells. Theαvβ3 integrin regulated up cdc2 expression of both mRNA and protein levels and activated MMP-2/9. Cdc2, a down stream effector of the αvβ3 integrin, associated with cyclin B2 regulated cell migration, active MMP-2/9 accelerate cell metastasis.