Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.     

Cloning and characterization of an ecdysone receptor cDNA from Locusta migratoria

It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.     

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.     

Increased expression of cyclin D1 in a murine mammary epithelial cell line induces p27kip1, inhibits growth, and enhances apoptosis

Cyclin D1 is frequently amplified and/or overexpressed in human breast cancer and several other types of cancer. To examine the role of cyclin D1 in normal mammary epithelial cells, in the present study we have overexpressed human cyclin D1 in the mouse mammary epithelial cell line HC11, using retrovirus-mediated transduction. We found that the cyclin D1 overexpresser clones displayed a decrease in saturation density, a decrease in anchorage-independent growth, an increased fraction of cells in the G(zero)-G1 phase, and increased expression of beta-casein, when compared to the control cells. The latter finding suggested that they were more differentiated. Furthermore, the cyclin D1 overexpressers displayed a marked increase in susceptibility to induction of apoptosis by serum withdrawal or by treatment with hydroxyurea or the protein kinase C inhibitors CGP 41251 and Ro31-8220. Thus, in some mammary epithelial cells, increased expression of cyclin D1 can inhibit growth, induce differentiation, and enhance apoptosis. These effects might be due, at least in part, to the fact that these derivatives displayed increased expression of the p27kip1 inhibitory protein.     

Phenotype of low proliferative potential is dominant in hybrids of normal human fibroblasts

A biochemical selection system was used to isolate hybrids after fusion of human diploid fibroblast clones of varying proliferative potential. The distribution of proliferative potentials of the hybrids resembled that of the parent with smaller proliferative potential. Therefore, the phenotype of limited division was dominant in hybrids. This dominance was associated with cells that could achieve seven or less divisions present in the parent populations. Terminally nondividing cells present in clones near the end of their proliferative potential differed from those present in clones that had ceased division (senescent), in that a higher percentage could be induced to divide at least once following fusion with a cell capable of division.     

The protein truncation test (PTT) as a method of detection for choroideremia mutations

The development of endocrine resistance in previously sensitive, estrogen receptor-positive breast cancers is a major limitation in the treatment of breast cancer. Because antiestrogens have a cell cycle-specific action on breast cancer cells and influence the expression and activity of several cell cycle-regulatory molecules, the development of aberrant cell cycle control mechanisms is a potential mechanism by which cells might develop resistance to antiestrogens. We postulated that overexpression of cyclin D1, which is a common feature of breast cancer, may confer antiestrogen resistance. We addressed this question in vitro by testing the ability of ectopic cyclin D1 overexpression to overcome the growth-inhibitory effects of tamoxifen and the pure steroidal antiestrogens, ICI 164384 and ICI 182780, in T-47D and MCF-7 human breast cancer cells. In cells stably transfected with a human cyclin D1 cDNA under the control of a metal-inducible metallothionein promoter, cyclin D1 expression was increased 2-4-fold following treatment with zinc. Despite the continued presence of antiestrogen, cyclin D1 induction resulted in the formation of active cyclin D1/Cdk4 complexes, concurrent hyperphosphorylation of the retinoblastoma protein, and entry into S phase of cells previously arrested in G1. Elevated cyclin D1 protein levels were first detected 3 h after treatment with zinc, and the proportion of cells in S phase began to increase 6 h later. The S-phase fraction increased 2-3-fold from 13 to 17% in cells treated with antiestrogen alone, to a peak of 33-38% 15 h after zinc treatment. Both the cyclin D1 protein level and the proportion of cells in S phase increased with increasing concentrations of zinc. We conclude that the ectopic overexpression of cyclin D1 reverses the growth-inhibitory effect of antiestrogens in estrogen receptor-positive breast cancer cells, providing a potential mechanism for clinical antiestrogen resistance.     

Increased expression of cyclin D1 and the Rb tumor suppressor gene in c-K-ras transformed rat enterocytes

Activating mutations in the c-K-ras gene occur in about 40% of human colorectal carcinomas, yet the role of this oncogene in tumorigenesis is not known. We have developed a model cell culture system to study this problem, utilizing the immortalized but non-tumorigenic epithelial cell line IEC18, originally derived from normal rat intestine epithelium. These cells were cotransfected with the drug resistance selectable marker tk-neo and the plasmid pMIKcys, which encodes a mini human c-K-ras gene (15 kb) containing a cysteine mutation at codon 12. Drug resistant clones were isolated. Clones which also expressed the activated c-K-ras gene displayed a transformed morphology, decreased doubling time, increased level of diacylglycerol, anchorage independent growth in soft agar and an aneuploid karyotype and they were also tumorigenic when injected into nude mice. These clones also displayed increased expression, at both the mRNA and protein levels, of cyclin D1 and Rb. These findings may be of clinical relevance since human colorectal tumors also frequently display increased expression of both cyclin D1 and Rb. This model system may be useful for understanding the role and interrelationship between activation of the c-K-ras oncogene and increased expression of cyclin D1 and Rb in colorectal tumorigenesis.     

SV40-transformed human cells in crisis exhibit changes that occur in normal cellular senescence

SV40 T antigen can induce senescent human diploid fibroblasts to synthesize DNA; however, the cells fail to go through mitosis. In this study, we examined the expression of the cdc2 and cyclin B genes, which are required for completion of mitosis, to determine whether defects in their expression occurred when SV40-transformed human cells entered the phase of crisis. If defects were observed it would indicate that immortalization by the virus involved reexpression of these genes. We found that the expression of cdc2 was unimpaired at both the RNA and protein levels, but that cyclin B expression was decreased in cells in crisis when compared with precrisis (mortal) and postcrisis (immortal) cells. Tritiated thymidine uptake demonstrated that the majority of cells in crisis were not actively cycling. Consistent with the latter observation we found that cyclin A, which is required for cells to traverse through S to G2, was downregulated in these cells. Since many of the results obtained with cells in crisis were similar to what is observed in normal human cells when they become senescent, we analyzed the expression of the genes fibronectin and sdi1 (a gene recently cloned from senescent cells that codes for an inhibitor of DNA synthesis). Both genes were overexpressed in cells during crisis, as is the case with senescent cells. The results are discussed in terms of the two-stage model previously proposed to explain the process of immortalization of human diploid fibroblasts by SV40.     

Reinitiation of DNA synthesis and cell division in senescent human fibroblasts by microinjection of anti-p53 antibodies

In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four- to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.     

Role of cyclin E and cyclin E-dependent kinase in mitogenic stimulation by cementum-derived growth factor in human fibroblasts

Cementum-derived growth factor (CGF) is a 14 kDa polypeptide sequestered in tooth cementum. It is an IGF-I like molecule that is weakly mitogenic to fibroblasts, but its mitogenic action is synergistically potentiated in the presence of epidermal growth factor (EGF) or serum. We have examined whether the CGF affects cyclin E levels and the activity of cyclin-dependent kinase (Cdk) associated with this cyclin, and whether these changes contribute to the synergism in mitogenic activity between CGF and EGF. Optimal DNA synthesis by serum-starved human gingival fibroblasts required the presence of CGF for 0-12 h and EGF for 0-3 h. Therefore, cells were serum starved for 48 h and then exposed to CGF, EGF, or CGF + EGF. Cells incubated with 10% fetal bovine serum (FBS) served as positive controls. At various time points after the addition of growth factors, cyclin E levels were examined by Western analysis. Cdk associated with cyclin E was immunoprecipitated with anti-cyclin E antibody and kinase activity was measured using H1 histone as substrate. Cyclin E and the H1 kinase activity levels increased after 8-12 h in cells exposed to CGF and in positive controls exposed to 10% FBS. They returned to basal level 4 h later in cells exposed to CGF alone, whereas in the presence of CGF + EGF and FBS they remained elevated for up to 20 h. The cyclin E levels did not increase in the presence of EGF alone. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 were barely detectable in these cells. Fibroblasts transfected with LXSN-cyclin E, a retroviral vector containing cyclin E cDNA, overexpressed cyclin E and their steady-state cyclin E-Cdk activity was higher than control cells. DNA synthesis by cyclin E overexpressing cells was higher, but optimal DNA synthesis by these cells required the presence of CGF and EGF. These results show that CGF action involves an increase in the levels of cyclin E and E-Cdk activity and that the higher levels are maintained in the presence of both CGF and EGF. They also indicate that sustained high cyclin E levels and Cdk2 activity during G1 phase are necessary, but not sufficient, for optimal mitogenic response in human fibroblasts.     

Regulated ectopic expression of cyclin D1 induces transcriptional activation of the cdk inhibitor p21 gene without altering cell cycle progression

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.     

Age-related changes in collagen gel contraction by cultured human lung fibroblasts resulting in cross-over of contraction curves between young and aged cells

We examined the effects of population doubling levels on collagen gel contraction by human lung fibroblasts (TIG-1). The sizes of gels at day 4 of culture, when the number of cells was the same as the initial number, were smaller with young cells than with aged cells. Therefore, retractive force had decreased with in vitro cellular aging. On the other hand, the lag time until gel contraction began became shorter with aging, resulting in the cross-over of contraction curves of young and aged cells. Morphological changes, such as pseudopodia protrusion, were suppressed in collagen gel. The surrounding collagen fibrils prevented young cells from moving more than aged cells. The weakened omnidirectional interaction with collagen fibrils on the entire surface of aged cells might result in an earlier occurrence of morphological change and, thereby, gel contraction.     

Changes in cyclins and cyclin-dependent kinases induced by DNA damaging agents in a human ovarian cancer cell line expressing mutated or wild-type P53

The expression of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors was evaluated in clones from a human ovarian cancer cell line transfected with a temperature-sensitive mutant of p53, after treatment with the anticancer agents doxorubicin (DX) and AMSA. The two drugs were selected on the basis of their activity in these clones, since AMSA is equally active in cells expressing mutated or wild-type (wt) p53, while DX was much less cytotoxic in cells expressing wt p53. In untreated cells, the expression of wt p53 induced an accumulation of cells in the G2 and perhaps also the G1 phase of the cell cycle. Concomitantly cyclin B1 and cdc2 increased. Cyclin E and particularly D1 levels were also raised by wt p53 expression. Treatment of mutated p53-expressing cells (SK23a cells kept at 37 degrees C) with DX or, more so, with AMSA, resulted in a strong accumulation of cyclin B1 and cdc2, in accordance with their ability to block cells in G2 phase of the cell cycle. Wt p53-expressing cells (SK23a cells kept at 32 degrees C) treated with the drugs showed an increase in p21 expression and consequently decreased kinase activity after immunoprecipitation with p21 antibodies. Cdc2-associated kinase activity was also reduced in these conditions. We could also observe a decrease in the percentage of cells in G1 and G2 phases and an accumulation of cells in S phase after both DX and AMSA. Cdk2, retinoblastoma, and p27 levels did not change significantly. Treatment with DX or AMSA caused similar effects, suggesting that p53-induced changes in cyclin, cdk, and cdk inhibitors after DNA damage are not responsible for the marked reduction in the cytotoxicity of DX we observed in wt p53-expressing cells.     

Cyclin D3 is rate-limiting for the G1/S phase transition in fibroblasts

D-type cyclins are induced in response to mitogens and are believed to control progression through the G1 phase of the cell cycle by activating their corresponding kinase partners (cyclin-dependent kinases). To investigate the function of individual D-type cyclins we have constructed rat fibroblast lines that allow controllable overexpression of a human cyclin D3 cDNA. Overexpression of cyclin D3 led to accelerated passage through G1 in actively proliferating cells with no effect on the overall population doubling time. In cells re-entering the division cycle from a quiescent state, cyclin D3 caused an even more dramatic advancement of S phase entry. Accelerated progression through G0/G1-to-S correlated with premature phosphorylation of the pRb tumor suppressor protein and its relatives, p107 and p130. We conclude that cyclin D3 can act as a rate-limiting G1 cyclin and that this effect involves, in part, the premature phosphorylation of critical substrates.     

A recombinant adenovirus expressing p27Kip1 induces cell cycle arrest and loss of cyclin-Cdk activity in human breast cancer cells

In order to elucidate the biochemical mechanisms by which the universal cyclin kinase inhibitor p27Kip1 regulates cell cycle progression in human breast cancer cells, a recombinant adenovirus expressing human p27 was constructed (Adp27). Upon infection of human breast cancer cells MDA-MB-231 and MCF-7 with Adp27, a high level of p27 expression was observed, and this resulted in a marked decrease in the proportion of cells in S-phase. In multiple cell lines, comparison of the cytotoxicity of Adp27 with another adenovirus vector expressing the related universal cyclin kinase inhibitor WAF1/Cip1 (AdWAF1), showed Adp27 to be markedly more (up to 56-fold) toxic than AdWAF1. DNA histograms showed Adp27 to cause a G1/S arrest at lower viral doses than AdWAF1. Analysis of cyclin dependent kinase activity following Adp27 infections showed decreased Cdk2 and cyclin B1-Cdc2 activity at lower viral doses when compared with AdWAF1. Adp27 is therefore potentially useful for studies of growth regulation and for gene therapy when growth inhibition is desired.     

TGF-beta1 effects on proliferation of rat intestinal epithelial cells are due to inhibition of cyclin D1 expression

Transforming growth factor-beta 1 (TGF-beta1) arrests intestinal epithelial cells (RIE-1 and IEC-6) in the G1 phase of the cell cycle and inhibits cyclin D1 expression. This report describes experiments designed to elucidate the mechanism of cyclin D1 inhibition and to determine whether inhibition of cyclin D1 expression is the cause, rather than the result, of TGF-beta1-mediated cell cycle arrest. TGF-beta1 inhibition of IEC-6 cell proliferation was associated with a decrease in the abundance of cyclin D1/Cdk4 complexes and a corresponding decrease in Cdk4-dependent phosphorylation of the retinoblastoma protein. Metabolic labeling studies indicated that TGF-beta1 inhibited cyclin D1 synthesis without altering the rate of cyclin D1 protein degradation. Cyclin D1 antisense oligonucleotides blocked serum-stimulated induction of cyclin D1 and DNA synthesis, whereas cyclin D1 sense oligonucleotides had no effect. RIE-1 cells were engineered to overexpress human cyclin D1 under the control of a tetracycline-repressible promoter. These cells entered S phase in the presence of TGF-beta1 only when human cyclin D1 was derepressed by the withdrawal of tetracycline. These data indicate that TGF-beta1 inhibits the synthesis of cyclin D1 in gut epithelial cells and that this inhibition is the cause, rather than the result, of TGF-beta1-mediated arrest of intestinal epithelial cell proliferation.