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Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have revolutionized the treatment of hormone-positive metastatic breast cancers (mBCs). They are currently established as standard therapies in combination with endocrine therapy as first- and second-line systemic treatment options for both endocrine-sensitive and endocrine-resistant mBC populations. In the first-line metastatic setting, the median progression-free survival for the three currently approved CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, with aromatase inhibitors is greater than 2 years (palbociclib 27.6 months; ribociclib 25.3 months; and abemaciclib 28.18 months). Although CDK4/6 inhibitors have significant clinical benefits and enable physicians to delay starting chemotherapy, they are expensive and can be associated with drug toxicities. Here, we have performed a systemic review of the reported molecular markers predictive of drug response including intrinsic and acquired resistance for CDK4/6 inhibition in mBC. The rapidly emerging molecular landscape is captured through next-generation sequencing of breast cancers (DNA with or without RNA), liquid biopsies (circulating tumor DNA), and protein analyses. Individual molecular candidates with robust and reliable evidence are discussed in more depth.

BackgroundLoss-of-function mutations of the retinoblastoma tumour suppressor RB1 are key drivers in cancer, with prominent involvement in the natural history of Osteosarcoma (OS). RB1 loss-of-function compromises genome maintenance in cells and hence could yield vulnerability to therapeutics targeting such processes.MethodWe assessed the response to Poly-ADP-Polymerase1/2 inhibitors (PARPi) in histiotype-matched cancer cell lines differing in RB1 status including an extended panel of OS lines, measuring viability, clonogenic activity and inhibition of xenograft growth in vivo. We used mutational signature analysis and RAD51 immunostaining to assess competence for homologous repair defect (HRd).ResultsWe report selective hypersensitivity to clinically-approved PARPi in OS lines with RB1 mutation, which extends to other cancer histiotypes and is induced in RB1-normal OS following engineered RB1 loss. PARPi treatment caused extensive cell death in RB1-mutated OS and extended survival of mice carrying human RB1-mutated OS grafts. Sensitivity in OS with natural or engineered RB1 loss surpassed that seen in BRCA-mutated backgrounds where PARPi are showing clinical benefit. PARPi sensitivity was not associated with loss of RAD51 recruitment and HRd-linked mutational signatures, which predict PARPi sensitivity in cancers with BRCA1/2 loss, but linked to rapid activation of replication checkpoint signalling with S phase transit critical for the death response observed.ConclusionOur work demonstrates that mutations in RB1 causes clinically relevant hypersensitivity to approved PARP1/2-targeting therapeutics and advocates PARP1/2 inhibition as a novel, genome lead strategy for RB1-mutated osteosarcoma.

To avoid replicative senescence or telomere-induced apoptosis, cancers employ telomere maintenance mechanisms (TMMs) involving either the upregulation of telomerase or the acquisition of recombination-based alternative telomere lengthening (ALT). The choice of TMM may differentially influence cancer evolution and be exploitable in targeted therapies. Here, we examine TMMs in a panel of 17 osteosarcoma-derived cell lines, defining three separate groups according to TMM and the length of telomeres maintained. Eight were ALT-positive, including the previously uncharacterized lines, KPD and LM7. While ALT-positive lines all showed excessive telomere length, ALT-negative cell lines fell into two groups according to their telomere length: HOS-MNNG, OHSN, SJSA-1, HAL, 143b, and HOS displayed subnormally short telomere length, while MG-63, MHM, and HuO-3N1 displayed long telomeres. Hence, we further subcategorized ALT-negative TMM into long-telomere (LT) and short-telomere (ST) maintenance groups. Importantly, subnormally short telomeres were significantly associated with hypersensitivity to three different therapeutics targeting the protein kinase ataxia telangiectasia and Rad3-related (ATR) (AZD-6738/Ceralasertib, VE-822/Berzoserib, and BAY-1895344) compared to long telomeres maintained via ALT or telomerase. Within 24 h of ATR inhibition, cells with short but not long telomeres displayed chromosome bridges and underwent cell death, indicating a selective dependency on ATR for chromosome stability. Collectively, our work provides a resource to identify links between the mode of telomere maintenance and drug sensitivity in osteosarcoma and indicates that telomere length predicts ATR inhibitor sensitivity in cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inhibitors of the Ataxia Telangiectasia and Rad3 related protein kinase (ATR) are increasingly entering clinical trials as single agents or in combination with inhibitors of poly (ADP-ribose) polymerases (PARPs), DNA-damaging chemotherapy or radiotherapy. However, and despite promise of broad utility and cancer selectivity, compelling results have remained an exception, underscoring the need to identify responsive patient population and tumour types. Based on in vitro testing in an extended cancer cell line panel we found that deleterious mutation of RB1 is significantly associated with hypersensitivity to multiple clinically relevant ATR inhibitors. Cancer cell lines with deleterious RB1 mutation displayed significantly enhanced cell death following ATR inhibitor treatment compared to a histotype matched cancer cell line groups with RB1 normal status and depletion of RB1 gene in RB1 normal cancer cell lines using siRNA, increased sensitivity to ATR inhibition accompanied by increased cells death. Deletion or deleterious mutation of RB1 is frequent in small cell lung cancers, cancers of neuroendocrine origin, and a considerable portion of triple negative breast and urinary tract cancers, cancers of the nervous system, sarcoma and chronic lymphatic leukemia. The data obtained document a link between RB1 status and ATR inhibitor sensitivity and argue that focus on cancer types with RB1 mutation or 13q14 involvement, where RB1 is encoded, should be considered in the design and analysis of trials involving ATR inhibitors. Citation Format: Caterina Mancusi, John Hartley, Sibylle Mittnacht. Tumor suppressor retinoblastoma status as a predictor of ATR inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 380.

The binding of RB to MDM2 is shown to be essential for RB to overcome both the antiapoptotic function of MDM2 and the MDM2-dependent degradation of p53. The RB-MDM2 interaction does not prevent MDM2 from inhibiting p53-dependent transcription, but the RB-MDM2 complex still binds to p53. Since RB specifically rescues the apoptotic function but not the transcriptional activity of p53 from negative regulation by MDM2, transactivation by wild-type p53 is not required for the apoptotic function of p53. However, an RB-MDM2-p53 trimeric complex is active in p53-mediated transrepression. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of RB in regulating the apoptotic function of p53.

Lack of detectable expression of p27kip1 cyclin dependent kinase inhibitor has previously been correlated with high degree of malignancy in human breast, colorectal, gastric and small cell lung carcinomas. Here we demonstrate that an inverse correlation between p27kip1 expression and tumour malignancy also exists in most types of human B cell lymphomas examined. A clear exception was Burkitt's lymphoma (BL), a highly malignant tumour which often expresses high levels of p27kip1. Analysis of p27kip1 derived from Burkitt's lymphoma cell lines expressing high levels of p27kip1, BL40 and BL41, in a cyclin E/cdk2 kinase inhibition assay demonstrated that p27kip1 is not permanently inactivated since heat treatment can restore the inhibitory activity of p27kip1. However, p27kip1 expressed in these two cell lines is largely sequestered in inactive complexes and we have no evidence that c-myc or Epstein-Barr virus are responsible for the sequestration of p27kip1 in these two cell lines although c-myc and EBV are two oncogenic agents often associated with Burkitt's lymphomas. Interestingly, we observed that high level p27kip1 expression often correlated with cyclin D3 overexpression both in vivo and in BL cell lines. The majority of p27kip1 in BL40 cells was complexed with cyclin D3 indicating that overexpressed cyclin D3 may at least be part of the sequestering activity for the inhibitory function of p27kip1. Furthermore, cyclinD3/cdk4 complex could sequester p27kip1 in a cyclin E/cdk2 kinase assay in vitro. Finally, we show that cyclin D3 transfected into an inducible p27kip1 cell line could overcome the G1 arrest mediated by p27kip1. These results argue that in addition to down-regulation of p27kip1 expression, some tumour cells can sequester and tolerate the antiproliferative function of p27kip1. They also suggest a novel role for the overexpression of D-type cyclins as one pathway allowing tumour cells to overcome the antiproliferative function of p27kip1.

BACKGROUND The tripartite motif family protein 27 (TRIM27) is a transcriptional repressor that interacts with, and attenuates senescence induction by, the retinoblastoma-associated protein (RB1). High expression of TRIM27 was noted in several human cancer types including breast and endometrial cancer, where elevated TRIM27 expression predicts poor prognosis. Here, we investigated the role of TRIM27 expression in cancer development. METHODS We assessed TRIM27 expression in human cancer using cancer profiling arrays containing paired tumor and normal cRNA (n = 261) as well as in murine skin cancer induced by 7, 12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA). We generated mice with disrupted expression of murine TRIM27 (Trim27(-/-)) and assessed their susceptibility to DMBA/TPA-induced skin tumor development compared with isogenic littermates (n = 26 mice per group). We assessed the effect of Trim27 loss on senescence propensity in mouse embryonic fibroblasts (MEFs) by quantifying cell proliferation alongside senescence markers (senescence-associated β-galactosidase [SA-β-gal] activity and hypertrophic cell morphology). The contribution of RB1 on senescence and cancer susceptibility (n > 20 mice per group) in Trim27(-/-) backgrounds was also assessed. Data were analyzed using the Student's t, χ(2), or log-rank test as indicated. All statistical tests were two-sided. RESULTS TRIM27 transcript levels are statistically significantly increased in common human cancers, including colon and lung, vs normal tissues (TRIM27 expression relative to ubiquitin: cancers vs normal tissues, mean = 0.59, 95% confidence interval [CI] = 0.55 to 0.63 vs mean = 0.46, 95% CI =0.43 to 0.49, P < .001) as well as in chemically induced mouse skin cancer compared with matched normal tissue (Trim27 expression relative to Gapdh control: tumor vs normal skin, mean = 4.2, 95% CI = 3.97 to 4.43 vs mean = 0.96, 95% CI = 0.69 to 1.2, P < .001). Trim27(-/-) mice (n = 14) were resistant to chemically induced skin cancer development (eight [57.2%] of 14 mice were tumor free) compared with Trim27(+/+) wild-type littermates (n = 13) (one [7.7%] of 13 mice was tumor free). Trim27(-/-) MEFs show enhanced senescence propensity in response to replicative (percentage of SA-β-gal-positive cells: Trim27(+/+) MEFs vs Trim27(-/-) MEFs, mean = 14.2%, 95% CI = 11.1% to 17.4% vs mean = 53.3%, 95% CI = 48.7% to 57.9%, P < .001) or oncogenic stress (percentage of SA-β-gal-positive cells: Trim27(+/+) MEFs + Ras vs Trim27(-/-) MEFs + Ras, mean = 24.0%, 95% CI = 19.9% to 28.1% vs mean = 37.3%, 95% CI = 32.2% to 42.4%, P < .05) compared with Trim27(+/+) MEFs. These responses were alleviated following inactivation of murine RB1 (Rb1). Furthermore, Trim27(-/-) mice are not protected from cancers arising as a consequence of Rb1 deletion (median survival: Trim27(-/-)Rb(+/-) vs Trim27(+/+)Rb(+/-), 14 vs 13 months; difference = 1.0 month, 95% CI = 0.5 to 1.6 months, P = .14). CONCLUSION TRIM27 expression is a modifier of disease incidence and progression relevant to the development of common human cancers and is a potential target for intervention in cancer.

Translocations of the retinoic acid receptor-alpha (RARalpha) locus with the promyelocytic leukemia zinc-finger (PLZF) or PML genes lead to expression of oncogenic PLZF-RARalpha or PML-RARalpha fusion proteins, respectively. These fusion oncoproteins constitutively repress RARalpha target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARalpha have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here, we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (poxvirus and zinc-finger, POZ) and DNA-binding (zinc-finger) domains. In addition, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction of Rb with PML or E2F1, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. These data are supported by chromatin immunoprecipitation analysis, which indicates that PLZF associates with the promoter region of CDC6, a known E2F/Rb target gene. Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F/Rb target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to function in stem cells and taken together these data suggest that interactions between PLZF and Rb could be important in stem cell biology.

Review on TRIM27 (tripartite motif containing 27), with data on DNA, on the protein encoded, and where the gene is implicated.

Summary The retinoblastoma tumor suppressor protein (Rb) affects gene transcription both negatively and positively and through this regulates distinct cellular responses. Although cell cycle regulation requires gene repression, Rb's ability to promote differentiation and part of its antiproliferative activity appears to rely on the activation of gene transcription. We present evidence here that the RET finger protein (RFP)/tripartite motif protein 27 (TRIM 27) inhibits gene transcription activation by Rb but does not affect gene repression. RFP binds to Rb and prevents the degradation of the EID-1 inhibitor of histone acetylation and differentiation. Furthermore, ablation of RFP in U2OS osteosarcoma cells augments a transcriptional program indicative of lineage-specific differentiation in response to Rb. These findings provide precedent for a regulatory pathway that uncouples different Rb-dependent activities and thus silences specific cellular responses to Rb in a selective way.

The retinoblastoma tumour suppressor protein (Rb) has come a long way since its initial discovery in 1986. Encoded by the first candidate tumour suppressor gene it has emerged a versatile and context-dependent modulator of cell behaviour. Its activity is managed by signalling networks sensing intra- and extracellular cues. These cues are relayed to hold or permit inactivation of Rb by phosphorylation. Loss or mutation of the retinoblastoma gene is rare in sporadic cancers but defects in the pathways that license inactivation of Rb are found in the majority of them, suggesting that loss of Rb control is central to tumour development and arguing that its reinstatement might reverse tumour formation. Furthermore, mouse models with engineered defects in the Rb-phosphorylating kinases provide evidence that moderation of Rb inactivation may be a strategy for the prevention of tumour formation. The rationale behind these arguments, their underlying molecular concepts and strategies towards therapeutic application will be discussed.

The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation.

The product of the retinoblastoma susceptibility gene, pRB, is a demonstrated substrate for the type 1 serine/threonine protein phosphatases (PP1). Curiously, there has been a paucity of data supporting the idea that phosphorylated pRB can be found in a complex with PP1. To more fully characterize the association between these two proteins, we utilized a PP1-affinity chromatography approach to increase our ability to capture from mammalian cell lysate populations of pRB capable of binding to PP1. Western blot analysis of the bound proteins indicates that both faster migrating, hypophosphorylated pRB, as well as slower migrating, hyperphosphorylated pRB can bind. Phosphorylated pRB binding was confirmed by immunoprecipitation of eluted 32P-labeled pRB. In addition, Western blotting of eluted proteins with pRB phosphorylated-site-specific antibodies revealed select phosphorylated forms of pRB binding to PP1. Similar binding studies performed with toxin-inhibited PP1 indicate that catalytic activity of PP1 is not required for pRB binding. The significance of this finding with respect to the functional importance of this interaction is discussed.

Cyclins are known effectors of cellular proliferation. While originally considered as the product of cellular genes, it is now clear that representatives of this class of proteins can be encoded by certain viruses. One of these viruses is HHV–8, a gamma herpesvirus implicated as a causative agent of Kaposi's Sarcoma and lymphomas in humans. The significance of the virally encoded cyclin proteins in viral propagation is as yet unclear. However, the fact that deregulation of cellular cyclin expression is a known event in tumour development suggests that the virally encoded cyclins could be part of a mechanism utilised by these viruses to induce tumour formation.

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.

Progression of cells into S phase is controlled by the retinoblastoma protein (pRB) and relies on the functional inactivation of this tumour suppressor in late G1 via protein phosphorylation. We provide evidence here that, besides controlling entry of cells into S phase, pRB can operate to inhibit S phase completion. Differential arrays of phosphorylation appear to regulate these different events, suggesting that cycle progression at these two stages of the cell cycle may be achieved via activation of distinct downstream pRB effector pathways. In agreement with this hypothesis, pRB's ability to prevent S phase entry, but not its ability to inhibit S phase completion, correlates with repression of E2F-regulated promoters. Furthermore, ectopic expression of E2F or the E2F-regulated cyclin E gene promote S phase entry in cells expressing phosphorylation-defective pRB but neither is sufficient to trigger completion of S phase. Our findings raise the possibility that pRB, in addition to its well-established role in controlling a checkpoint in late G1, could be involved in the control of a further checkpoint operating during S phase and that implementation of this checkpoint relies on an as yet unidentified pRB effector distinct from E2F.

Two opposing enzymatic reactions control the activity of the retinoblastoma tumour suppressor protein, pRB. Phosphorylation inactivates pRB's ability to sequester miscellaneous cellular proteins, mostly involved in regulating gene transcription, whereas pRB dephosphorylation restores this ability. For some time now it has been suspected that members of the cyclin/cyclin-dependent kinase (cyclin/cdk) family mediate pRB inactivation. Recent results indicate that pRB phosphorylation is not executed by single kinase but by a combination of cyclin/cdks, each one phosphorylating a subset of pRB's phosphorylation sites. The different kinases appear to be activated by growth factors through distinct signal transduction pathways. This lends itself to an attractive model whereby pRB phosphorylation may constitute an integration point for these signalling pathways, perhaps allowing cell cycle progression only when concurrent activation of these signalling pathways has been achieved.

The growth suppressive activity of the retinoblastoma tumour suppressor protein is controlled by cell cycle dependent phosphorylation. However, while many in vivo phosphorylation sites have been mapped, the identities of those residues whose phosphorylation is regulated remain elusive. We have mapped the epitopes of three independent monoclonal antibodies that recognise a distinction between differentially phosphorylated pRB sub-populations. All three antibodies recognise an identical epitope which encompasses an essential serine positioned within a consensus site for proline directed kinase phosphorylation. We provide evidence that this residue, serine 608 of pRB, is an authentic phosphorylation site that can be phosphorylated in vitro by cyclin A-CDK2 and cyclin D1-CDK4 kinases but not by cyclin E-CDK2 kinase or the mitogen activated kinase ERK2. Phosphorylation at this residue seems to be cell cycle regulated, occurring prior to entry into the S phase.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.

Loss of retinoblastoma protein (RB1) function is a major driver in cancer development. We have recently reported that, in addition to its well-documented functions in cell cycle and fate control, RB1 and its paralogs have a novel role in regulating DNA repair by non-homologous end joining (NHEJ). Here we summarize our findings and present mechanistic hypotheses on how RB1 may support the DNA repair process and the therapeutic implications for patients who harbor RB1-negative cancers.

Background The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1. Results The requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal. Conclusion The work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct.

The Kaposi's Sarcoma associated Herpes virus (KSHV) encodes two genes with the potential to affect the activity of the retinoblastoma protein (Rb). Open reading frame (orf) 72 encodes a D type cyclin (kcyc) that can elicit p16INK4a resistant cdk activity and orf73 encodes the latency associated nuclear antigen (LNA) that can bind Rb and neutralize E2F regulation. This indicates that, like papilloma and adenovirus associated malignancies, those associated with KSHV are defective with respect to their Rb pathway. To address this we investigated whether KSHV associated primary effusion lymphoma (PEL) derived cell lines are resistant to growth inhibition by p16INK4a. We provide evidence that ectopic expression of p16INK4a in these cells causes an Rb dependent G1 cell cycle block. Importantly, endogenous p16INK4a expression is not detected in six PEL derived cell lines and four primary PEL samples and examination of the p16INK4a locus shows deletion in two out of six and hypermethylation in four out of six PEL lines. Treatment of the latter with the demethylating agent 5'-aza-2' deoxycytidine leads to re-expression of p16INK4a protein. Taken together these results suggest that p16INK4a loss may be a cellular change frequently associated with PEL. They furthermore argue that despite the presence of KSHV DNA and expression of a latent gene program Rb function is intact in PEL.

pRb is dephosphorylated at mitotic exit by the type 1 serine/threonine protein phosphatases (PP1). Here we demonstrate for the first time that mitotic pRb dephosphorylation is a sequential, temporally-regulated event. We also provide evidence that the three mammalian isoforms of PP1, alpha, gamma-1, and delta, differ in their respective preferences for site-specific pRb dephosphorylation and that the mitotic and G(1) PP1-isoform counterparts exhibit differential activities towards mitotic pRb. Finally, the physiological relevance of the striking contrast between the patterns of Thr821 and Thr826 dephosphorylation, sites known to be important for disrupting binding of LXCXE-containing proteins to pRb, is addressed.

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) is a novel gammaherpesvirus implicated in the cause of Kaposi's sarcoma and certain malignancies of lymphatic origin. One of the candidate genes possibly involved in promoting tumor development is an open reading frame (ORF) with sequence similarity to human type D cyclin genes. This cyclin-like gene, when expressed in tissue culture cells, promotes phosphorylation and inactivation of the retinoblastoma tumor suppressor protein and thereby may result in deregulation of cell division control. We report here the biochemical characterization of this cyclin (KSHV-cyc) and the kinase activity that it elicits upon expression in tissue culture cells. We demonstrate that the kinase activity associated with KSHV-cyc is sensitive to the cdk inhibitor p27 (KIP) and due to activation of cdk6. However, in contrast to cdk6 activated by cellular type D cyclins, the cdk6 activated by KSHV-cyc is capable of phosphorylating not only the retinoblastoma protein but also histone H1. This finding implies that activation by KSHV-cyc alters the substrate preference of this cdk. This may have important physiological consequences in that the kinase activity triggered by this viral cyclin may abrogate cell cycle checkpoints in addition to those targeted by cellular cyclin D-cdk6 kinase.

There is increasing evidence for the therapeutic effectiveness of Interferon-alpha (IFN-alpha) in malignant diseases. However, the antitumor mechanisms of IFN-alpha are not known. Using two examples, hairy Cell leukemia (HCL) and renal cell cancer (RCC), it is shown that the requirements for successful IFN-alpha therapy of HCL and RCC are different. In HCL low doses of IFN-alpha are sufficient to treat the disease. The reduction of hairy cells in peripheral blood is detectable within the first week of treatment. The endogenous IFN-alpha production in these patients is impaired as demonstrated by the lack of IFN-alpha induction and by low levels of 2-5 oligoadenylate synthetase in peripheral blood mononuclear cells. A possible reason for deficient endogenous IFN-alpha production is the lack of monocytes in HCL patients. It is likely that therapy with low doses of IFN-alpha substitutes for the endogenous IFN-alpha deficiency. In RCC comparatively high doses of IFN-alpha are necessary for a clinical response. There may be differences between the effectiveness of natural and recombinant alpha interferons. High doses given within a week seem to be more important than high single doses, which therefore suggests the need of daily treatment. Responses of RCC to IFN-alpha therapy are usually seen several months after the beginning of therapy. These differences in the effectiveness of IFN-alpha therapy for HCL and RCC suggest that IFN-alpha acts differently in the treatment of each disease.