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Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea-induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15-fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans.

In the present study we have characterized eight human esophageal squamous carcinoma cell lines for levels of expression of cyclins D1, E, A and B1; CDKs 1, 2 and 4; the CDK inhibitors p16INK4, p21WAF1 and p27KIP1; the retinoblastoma (Rb) protein; and in vitro CDK2- and CDK4-associated kinase activity; and also compared the growth properties of these cell lines. The level of the cyclin D1 protein varied by over 30-fold amongst the eight cell lines. The high level in two cell lines was associated with amplification of this gene, but in three cell lines it was due to post-transcriptional events. Amongst the eight cell lines there was a significant correlation between the levels of cyclin D1, Rb and p27KIP1 proteins, and CDK4-associated kinase activity. Furthermore, when an exogenous cyclin D1 cDNA was over-expressed in the EC109 cell line by transfection, this led to increased expression of both Rb and p27KIP1. There was, however, no correlation between the level of cyclin D1 expression and the cell doubling times, duration of the G1 phase, or colony-forming efficiency in agar. Two of the cell lines displayed a high level of the cyclin E protein, low levels of cyclin D1, lacked expression of the Rb protein and expressed high levels of the p16INK4 protein. One of these cell lines displayed amplification of the latter gene. There was no correlation between the levels of cyclins E or A and in vitro CDK2 kinase activity, but CDK2 kinase activity was inversely correlated with the duration of the G1 phase of the cell cycle. Taken together, these studies indicate marked heterogeneity in the expression of cell cycle-related proteins amongst a series of esophageal carcinoma cell lines. The correlation between the levels of the cyclin D1, Rb and p27Kip1 proteins suggest the existence of a homeostatic feedback loop between positive and negative acting components of the cell cycle machinery.

Since PKC epsilon functions as an oncogene when stably overexpressed in R6 rat fibroblasts (Cacace et al. 1993) in the present study we examined whether transformed R6-PKC epsilon cells display abnormalities in the expression of specific early response and cyclin genes. When vector control and R6-PKC epsilon cells were starved of serum for 72 h they arrested in G0/G1 and showed passage through the cell cycle at similar rates after subsequent stimulation with 10% fetal calf serum plus TPA. In PKC epsilon cells, induction of cyclin D1 protein was markedly reduced, and that of cyclin A was slightly reduced when compared to control cells. Northern blot analyses indicated that decreased expression of cyclin D1 and A protein in PKC epsilon cells is due to translational or post-translational effects. A study of early response gene expression in PKC epsilon cells indicated that there was a marked reduction in the expression of c-fos mRNA but not in c-jun or c-myc mRNAs. The marked decreases in cyclin D1 and c-fos expression seen in PKC epsilon cells were not seen in R6 cells that overexpress PKCs alpha or beta. These findings suggest that PKC epsilon cells bypass certain normal signal transduction and cyclin-controlled pathways involved in cell proliferation.