The kinetics of simian virus 40-induced progression of quiescent cells into S phase depend on four independent functions of large T antigen.

Microinjection of purified simian virus 40 large-T-antigen protein or DNA encoding T antigen into serum-starved cells stimulates them to re-enter the cell cycle and progress through G1 into the S phase. Genetic analysis of T antigen indicated that neither its Rb/p107-binding activity nor its p53-binding activity is essential to induce DNA synthesis in CV1P cells. However, T antigens bearing missense mutations that inactivate either activity induced slower progression of the cells into the S phase than did wild-type T antigen. Inactivation of both activities resulted in a T antigen essentially unable to induce DNA synthesis. Missense mutations in either the DNA-binding region of the N terminus also impaired the ability of full-length T antigen to stimulate DNA synthesis in CV1P cells. The wild-type kinetics of cell cycle progression were restored by genetic complementation after coinjection of plasmid DNAs encoding different mutant T antigens or coinjection of purified mutant T-antigen proteins, suggesting that the four mitogenic functions of T antigen are independent. The maximal rate of induction of DNA synthesis in secondary primate cells and established rodent cell lines required the same four functions of T antigen. A model to explain how four independent activities could cooperate to stimulate cell cycle progression is presented.     

DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.     

A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary

A novel cell cycle blocking agent profoundly suppressed the proliferation of mitogen-stimulated T lymphocytes. The carboxythiazole derivative arrested cells in the G1 phase of the cell cycle but did not inhibit the induction of cell surface receptors for either interleukin-2 or transferrin. The uncoupling of transferrin receptor expression from DNA synthesis indicated that a previously undefined restriction point in the cell cycle has been identified which occurs after transferrin receptor expression in late G1 and just prior to the initiation of DNA replication in S phase. T cells incubated in an inhibitory dose of the carboxythiazole derivative resumed cell cycle progression subsequent to its removal, indicating that the compound reversibly arrests cells at the late G1 restriction point. In contrast to other techniques which have been inefficient in achieving T cell synchronization, T cells released from the block mediated by the carboxythiazole compound progress through S phase with a considerable degree of synchrony.     

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.     

Growth inhibition by TGF-beta linked to suppression of retinoblastoma protein phosphorylation

The growth-suppressive function of the retinoblastoma gene product, RB, has been ascribed to the underphosphorylated RB form that prevails during G1 phase in the cell cycle. We show that addition of the paracrine growth inhibitor transforming growth factor beta 1 (TGF-beta 1) to Mv1Lu lung epithelial cells in mid to late G1 prevents phosphorylation of RB scheduled for this cell cycle stage and arrests cells in late G1. Expression of SV40 T antigen, a transforming protein that binds underphosphorylated RB, does not block the effect of TGF-beta 1 on RB phosphorylation but greatly reduces the growth-inhibitory response to TGF-beta 1. TGF-beta 1 and RB appear to function in a common growth-inhibitory pathway in which TGF-beta 1 acts to retain RB in the underphosphorylated, growth-suppressive state.     

Hypophosphorylation of the RB Protein in S and G2 as Well as G1 during Growth Arrest

The RB tumor suppressor protein is a cell cycle regulator, where hypophosphorylated RB is associated with G1/0 arrest and its cyclin-dependent phosphorylation in G1 allows progression from G1 to S. The present report shows that in human leukemia cells induced to undergo growth arrest with sodium butyrate or DMSO, hypophosphorylation of the RB protein is not G1 restricted and also occurs in S and G2/M cells as well as in G1 cells when growth is inhibited. While all of the RB protein in G1/0 cells is hypophosphorylated, residual cells in S and G2 have significant detectable amounts of hypophosphorylated RB as well as still hyperphosphorylated RB protein. Thus RB hypophosphorylation can be induced in S and G2 as well as the G1 phase. The results show that growth retardation in other than the G1 phase is associated with occurrence of hypophosphorylated RB. RB may thus have a broader capability to inhibit proliferation than just in G1.     

The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T

Synchronized monkey cells pulse-labeled with [35S]-methionine and chased for various lengths of time were extracted, and immunoprecipitations were performed using monoclonal antibodies directed against the retinoblastoma protein (RB) and SV40 T antigen (T). By following a discrete population of these two proteins through the cell cycle, the following information was obtained. RB, which is wholly unphosphorylated in G1, became phosphorylated at the beginning of S and remained phosphorylated through S and G2. RB was, then, completely dephosphorylated between the end of G2 and the beginning of G1. Second, while all of the detectable unphosphorylated RB can be found complexed with T, these complexes present during G1 dissociated in S and reformed again in M or early G1. Finally, T molecules appeared to oligomerize prior to binding RB. Thus, complex formation between T and RB may be regulated in part by the cell cycle-dependent phosphorylation and dephosphorylation of RB and by the quaternary structure of T.     

A UV-responsive G2 checkpoint in rodent cells.

We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed.     

Retinoblastoma tumor suppressor protein signals through inhibition of cyclin-dependent kinase 2 activity to disrupt PCNA function in S phase

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G(1) and S-phase progression. While RB-mediated G(1) inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition.     

Differential requirements for ras and the retinoblastoma tumor suppressor protein in the androgen dependence of prostatic adenocarcinoma cells.

Prostate cells are dependent on androgen for proliferation, but during tumor progression prostate cancer cells achieve independence from the androgen requirement. We report that androgen withdrawal fails to inhibit cell cycle progression or influence the expression of cyclin-dependent kinase (CDK)/cyclins in androgen-independent prostate cancer cells, indicating that these cells signal for cell cycle progression in the absence of androgen. However, phosphorylation of the retinoblastoma tumor suppressor protein (RB) is still required for G1-S progression in androgen-independent cells, since the expression of constitutively active RB (PSM-RB) or p16ink4a caused cell cycle arrest and mimicked the effects of androgen withdrawal on downstream targets in androgen-dependent LNCaP cells. Since Ras is known to mediate mitogenic signaling to RB, we hypothesized that active V12Ras would induce androgen-independent cell cycle progression in LNCaP cells. Although V12Ras was able to stimulate ERK phosphorylation and induce cyclin D1 expression in the absence of androgen, it was not sufficient to promote androgen-independent cell cycle progression. Similarly, ectopic expression of CDK4/cyclin D1, which stimulated RB phosphorylation in the presence of androgen, was incapable of inactivating RB or driving cell cycle progression in the absence of androgen. We show that androgen regulates both CDK4/cyclin D1 and CDK2 complexes to inactivate RB and initiate cell cycle progression. Together, these data show that androgen independence is achieved via deregulation of the androgen to RB signal, and that this signal can only be partially initiated by the Ras pathway in androgen-dependent cells.     

RB-dependent S-phase response to DNA damage

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.     

Radiofrequency electromagnetic fields do not alter the cell cycle progression of C3H 10T and U87MG cells.

The effects of exposure to radiofrequency electromagnetic fields (RF EMFs) on cell cycle progression of mouse fibroblasts C3H 10T(1/2) and human glioma U87MG cells were determined by the flow cytometric bromodeoxyuridine pulse-chase method. Cells were exposed to a frequency-modulated continuous wave at 835.62 MHz or a code division multiple access RF EMF centered on 847.74 MHz at an average specific absorption rate of 0.6 W/kg. Five cell cycle parameters, including the transit of cells through G(1), G(2) and S phase and the probability of cell division, were examined immediately after the cells were placed in the fields or after they had been kept in the fields for up to 100 h. The only significant change observed in the study was that associated with C3H 10T(1/2) cell cultures moving into plateau phase toward the later times in the long-exposure experiment. No changes in the cell cycle parameters were observed in cells exposed to either mode of RF EMFs when compared to sham-exposed cells in either of the cell lines studied during the entire experimental period. The results show that exposure to RF EMFs, at the frequencies and power tested, does not have any effect on cell progression in vitro.     

C-terminal phosphorylation controls the stability and function of p27kip1

Entry of cells into the cell division cycle requires the coordinated activation of cyclin-dependent kinases (cdks) and the deactivation of cyclin kinase inhibitors. Degradation of p27kip1 is known to be a central component of this process as it allows controlled activation of cdk2-associated kinase activity. Turnover of p27 at the G1/S transition is regulated through phosphorylation at T187 and subsequent SCF(skp2)-dependent ubiquitylation. However, detailed analysis of this process revealed the existence of additional pathways that regulate the abundance of the protein in early G1 and as cells exit quiescence. Here, we report on a molecular mechanism that regulates p27 stability by phosphorylation at T198. Phosphorylation of p27 at T198 prevents ubiquitin-dependent degradation of free p27. T198 phosphorylation also controls progression through the G1 phase of the cell cycle by regulating the association of p27 with cyclin-cdk complexes. Our results unveil the molecular composition of a pathway, which regulates the abundance and activity of p27kip1 during early G1. They also explain how the T187- and the T198-dependent turnover systems synergize to allow cell cycle progression in G1.