New functional activities for the p21 family of CDK inhibitors

The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors p21(CIP), p27(KIP), and p57(KIP2) all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K(a), respectively, mostly because of a decrease in K(off). At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a G(0) arrest. In agreement with our other data, cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.     

Degradation of p27(Kip) cdk inhibitor triggered by Kaposi's sarcoma virus cyclin-cdk6 complex

The Kaposi's sarcoma-associated human herpesvirus 8 (KSHV/HHV8) encodes a protein similar to cellular cyclins. This cyclin is most closely related to cellular D-type cyclins, but biochemically it behaves atypically in various respects. Complexes formed between the viral cyclin and the cyclin-dependent kinase subunit, cdk6, can phosphorylate a wider range of substrates and are resistant to cdk inhibitory proteins. We show here that the KSHV-cyclin-cdk6 complex phosphorylates p27(Kip) on a C-terminal threonine that is implicated in destabilization of this cdk inhibitor. Expression of the viral cyclin in tissue culture cells overcomes a cell cycle block by p27(Kip). However, full cell-cycle transit of these cells appears to depend on C-terminal phosphorylation of p27(Kip) and seems to involve transactivation of other cellular cyclin-dependent kinases. A p27(Kip)-phosphorylating cdk6 complex exists in cell lines derived from primary effusion lymphoma and in Kaposi's sarcoma, this indicating that virally induced p27(Kip) degradation may occur in KSHV-associated tumours.     

The CDK inhibitor, p27Kip1, is required for IL-4 regulation of astrocyte proliferation

IL-4 is a pleiotrophic cytokine that has been shown to affect cells of the central nervous system. We have demonstrated that IL-4 inhibits DNA synthesis and proliferation in human astroglia expressing IL-4 receptors. In this study, we sought to identify mechanisms that could account for the antimitogenic effects of IL-4. Epidermal growth factor (EGF)-stimulated human astroglia were arrested in G1 phase by IL-4, even though IL-4 stimulated levels of the G1 cyclins, D1 and E. Histone H1 kinase activity of cdk2 immunoprecipitates, however, was sharply reduced by IL-4; impairment of kinase activity was also evident in cyclin E immunoprecipitates, which contained evidence of hypophosphorylated (inactive) cdk2 product. Reduced cyclin E-associated cdk2 activity was not due to impaired cyclin-dependent kinase-activating kinase (CAK) activity, which was unaffected by IL-4. Inactive cyclin E/cdk2 complexes from IL-4 + EGF-treated cells contained, however, strikingly elevated p27Kip1 cdk inhibitor. Elevated p27 was also detectable in whole cell lysates after 24 and 48 h of IL-4 treatment; by 72 h, p27 was no longer elevated. Pretreatment with antisense but not mismatch p27 oligonucleotides attenuated the inhibitory effects of IL-4 on DNA synthesis and histone kinase activity of cyclin E/cdk2 complexes. Antisense p27 also abrogated IL-4-mediated elevation of p27 in whole cell lysates and cyclin E/cdk2 complexes. These findings demonstrate that IL-4 regulates the cell cycle machinery of astroglial cells via a p27Kip1 braking mechanism.     

Expression of G1 cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors in androgen-induced prostate proliferation in castrated rats

Androgen induces prostate cell proliferation in the castrated rat. We hypothesized that G1 cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors mediate this cellular response to mitogenic signals. In this study, induction of cyclins D1, D2, D3, E, and cdks 2, 4, and 6 expression was observed at various time points during testosterone replacement in the ventral prostate of castrated rats. The induction followed prostate epithelium proliferation, which peaked at 48 h and decreased at 120 h during the treatment. The study of cyclin/cdk complex formation revealed that more cyclin D1/cdk4 and cyclin D1/cdk6 complexes were formed at 48 h than at 120 h of treatment, but cyclin D1/cdk2 complexes remained the same. Furthermore, both hyperphosphorylated and hypophosphorylated forms of Rb were detected at 48 h, but only the hypophosphorylated form was detected at 120 h of treatment. p21Cip1, which was very abundant in the ventral prostate of castrated and intact rats, was not detected when the prostate started proliferation and increased gradually as proliferation decreased during the androgen treatment. Meanwhile, p27Kip1 dramatically increased after androgen treatment, and the induction levels were less at the peak of prostate proliferation and higher when proliferation was low. The results presented here suggest that expression of G1 cyclins and their related kinases and kinase inhibitors are well regulated after androgen replacement in the ventral prostate of castrated rats. The cooperation between these cell cycle regulators leads to a well-controlled prostate regeneration.     

Neuronal expression of cycline dependent kinase inhibitors of the INK4 family in Alzheimer's disease

Neurodegeneration and cell death in Alzheimer's disease might be associated with aberrant proliferative mechanisms and activation of cell-cycle related events. We reported previously on the elevated expression of the cyclin dependent kinase inhibitor p16INK4a in Alzheimer's disease closely associated with neurofibrillary degeneration. In the present study, we demonstrate that other members of the INK4-family of cyclin dependent kinase inhibitors such as p15INK4b, p18INK4c and p19INK4d that bind directly to cdk4/6 or to complexes of cdk4/6 with D-type cyclins are all elevated. In contrast, no indication of altered expression of the cyclin dependent kinase inhibitors p21Cip1 and p27Kip1 were observed. Inhibitors of the INK4-family were strongly expressed in tangle-bearing neurones and neuritic components of plaques. A much lower expression was also seen in astrocytes. These findings add further evidence to the suggestion that a dysfunction of cell cycle regulation is of critical importance in the pathomechanism of Alzheimer's disease.     

CD40-mediated induction of p21 accumulation in resting and cycling B cells

The accumulation of G1 cell cycle-related proteins by resting or cycling B cells stimulated with B cell antigen receptor (BCR)- and T helper (Th) cell-derived signals is documented. Resting B cells constitutively express cyclin dependent kinase (cdk)4, cdk2 and the cyclin dependent kinase inhibitor (CKI), p27. The initiation of optimal proliferation with F(ab')2 anti-mu plus paraformaldehyde-fixed CD40 ligand-baculovirus-infected Sf9 cells (CD40L/Sf9 cells) increases accumulation of both cdk4 and cdk2 while decreasing p27 levels. B cells express cyclin D2 early during cycle progression, while cyclin D3 and E are not expressed until 18 h poststimulation and cyclin A by 24 h poststimulation. Cycling B cells express heightened levels of all these cyclins and cdks. Although neither BCR- nor CD40-mediated signals appreciably alter cycling B cell accumulation of cyclins D2, cdk4 and cdk2, the absence of BCR-derived signals results in a decreased accumulation of cyclins D3 and E. Finally, CD40-mediated signals induce resting B cells to accumulate the CKI, p21, while cycling B cells require both BCR- and CD40-mediated signals to maintain increased expression of p21. Thus, a Th cell-derived signal may impact upon both resting and cycling B cell cycle progression, at least in part, by regulating the accumulation of p21. The functional consequences of p21 accumulation as cells enter and move through the cell cycle are discussed.     

TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.     

Cyclin D- and E-dependent kinases and the p57(KIP2) inhibitor: cooperative interactions in vivo

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.     

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.     

Absence of cyclin D/cdk complexes in cells lacking functional retinoblastoma protein

Cyclins D1, D2 and D3 are thought to function in the G1 phase of the cell division cycle by regulating the activity of cyclin-dependent protein kinases. All three D-type cyclins can be shown to associate with two specific kinases, cdk4 and cdk6, providing at least six possible combinations. To establish whether different cell types require different subsets of these complexes and whether they are altered in tumours where D-cyclin expression is perturbed, we surveyed a series of tumour cell lines and compared them where possible to non-tumorigenic counterparts. Although complexes involving cdk4 or cdk6 were readily observed in many of the cell lines, no complexes were detectable in human cells harbouring DNA tumour virus oncoproteins or in which the retinblastoma gene product (pRb) is mutated or missing. These data suggest that as well as being a potential substrate for D-cyclin-kinases, functional pRb contributes to the formation or stability of the complexes, at least in human cells.     

G1 arrest of U937 cells by onconase is associated with suppression of cyclin D3 expression, induction of p16INK4A, p21WAF1/CIP1 and p27KIP and decreased pRb phosphorylation

Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.     

Pramipexole reduces reactive oxygen species production in vivo and in vitro and inhibits the mitochondrial permeability transition produced by the parkinsonian neurotoxin methylpyridinium ion

CeReS-18 is a unique negative regulator of cell proliferation with a wide array of target cells. To elucidate the mechanism by which CeReS-18 mediates cell growth inhibition, the possibility that CeReS-18 alters the function of G1 cyclins and their respective cyclin-dependent kinases (cdks) has been examined in mouse fibroblasts (Swiss 3T3) synchronized by CeReS-18. We show here that cyclin D-associated cdk activity is significantly inhibited in the CeReS-18-treated cells. Corresponding to the inhibited cdk function, we demonstrate a low expression of cyclin D in mid G1 determined by Western blot analysis, and cyclin D was greatly reduced in the immunocomplex recovered with antibody to cdk4 and cdk6. Previously, we have shown that the retinoblastoma susceptibility gene product (pRb), a key substrate of cyclin D-cdk complex, was maintained in the hypophosphorylated state in the CeReS-18-inhibited cells. We conclude here that cyclin D/cdk4,6/pRb is the major pathway by which CeReS-18 mediates cell cycle arrest.     

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.     

"U" curve association of blood pressure and mortality in hemodialysis patients. Medical Directors of Dialysis Clinic, Inc

An immunosuppressant Rapamycin (Rap) has been reported to cause G1 arrest by inhibiting p70 S6 kinase and G1 cyclin/cdks kinase activities when added to quiescent cells with mitogens. However, antiproliferative effects of Rap on exponentially growing cells have been poorly investigated. We examined the intracellular events after the treatment of Rap in exponentially growing T cells and found that Rap upregulated a cdks inhibitor, p27Kip1 at both mRNA and protein levels in Rap-sensitive cells. Antiproliferative effect of Rap was mainly ascribed to the inhibition of cyclin E/cdk2 kinase activity through the formation of cyclin E/cdk2-p27Kip1 complex rather than inhibition of p70 S6 kinase activity. Furthermore, we showed that Rap-sensitive cells with elevated p27Kip1 expression lost sensitivity to Rap when antisense p27Kip1 was introduced, which indicates that the basal level of p27Kip1 is one of the limiting factors that determine the sensitivity to Rap in already cycling cells. These data suggest the presence of a putative threshold level of p27Kip1 at late G1 phase in already cycling cells. Rap may cause G1 arrest by upregulating the amount of p27Kip1 beyond the threshold in some Rap-sensitive cells that are exponentially growing.     

Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells

Loss of attachment to an extracellular matrix substrate arrests the growth of untransformed cells in the G1 phase. This anchorage-dependent cell cycle arrest is linked to increased expression of the p21Cip1 (p21) and p27Kip1 (p27) cyclin-dependent kinase inhibitors. The result is a loss of cdk2-associated kinase activity, especially that of cyclin E-cdk2. The levels of p21 and p27 are also upregulated in unattached transformed cells, but cyclin E-cdk2 activity remains high, and the cells are able to grow in an anchorage-independent manner. Increased expression of cyclin E and cdk2 appears to be partially responsible for the maintenance of cyclin E-cdk2 activity in transformed cells. To explore further the regulation of cyclin E-cdk2 in transformed cells, we have analysed the subcellular distribution of cyclin-cdk complexes and their inhibitors in normal human fibroblasts, their transformed counterparts, and in various human tumor cell lines. In substrate-attached normal fibroblasts, cyclin E and cdk2 were exclusively in the nuclear fraction, associated with one another. When normal fibroblasts were detached and held in suspension, cyclin E-cdk2 complexes remained nuclear, but were now found associated with the p21 and p27 cdk inhibitors and lacked histone H1 phosphorylating activity. In contrast, the transformed fibroblasts and tumor cells, which are anchorage-independent, had more than half of their cyclin E, cdk2, p21 and p27 in the cytoplasmic fraction, both in attached and suspended cultures. The cytoplasmic p21 and p27 were bound to cyclin E-cdk2, as well as to complexes containing cyclin A and cyclin D. The nuclear cyclin E-cdk2 complexes from the transformed cells grown in suspension contained only low levels of p21 and p27 and had histone H1 kinase activity. Thus, at least three mechanisms contribute to keeping cyclin E-cdk2 complexes active in suspended anchorage-independent cells: cyclin E and cdk2 are upregulated, as reported previously, cdk inhibitors are sequestered away from the nucleus by cytoplasmic cyclin-cdk complexes, and the binding of the inhibitors to nuclear cyclin E-cdk2 complexes is impaired.     

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 --> S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.     

Cyclin D1 and retinoblastoma protein expression in Kaposi's sarcoma

Cyclins are implicated in the induction and control of the cell cycle. Cyclin D1 regulates G1-phase progression by phosphorylation of the retinoblastoma protein (pRb). The Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV) contains and transcribes an open reading frame with sequence similarities to cellular D-type cyclins. The KSHV-cyclin protein is associated with kinase activity capable of phosphorylating pRb in vitro. Here, we study for the first time the endogenous cyclin D1 and Rb protein expression in Kaposi's sarcoma (KS) tissue. Twenty-four consecutive biopsies of AIDS-related (n=21) and classical (n=3) KS were studied by immunohistochemistry with monoclonal antibodies against cyclin D1 and pRb. We detected cyclin D1 in 1 of 13 patch/plaque stage, in 4 of 5 nodular stage and in 3 of 6 visceral KS lesions. By Western blot analysis, this cellular cyclin D1 monoclonal antibody did not cross-react with the purified KSHV-cyclin protein. The pRb was consistently detected in 24 of 24 KS lesions. In summary, early KS lesions rarely have detectable expression of endogenous cyclin D1. Advanced and disseminated KS lesions tend to have overexpression of endogenous cyclin D1. Therefore, cellular cyclin D1 expression appears to correlate with tumor progression in KS. The endogenous cyclin D1 is antigenically distinct from the KSHV-cyclin homolog. The pRb, which may serve as a substrate for KSHV-cyclin, is found in all KS lesions examined.