CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1-induced T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new therapeutic approaches are needed. We showed previously that cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy.

Melanocytic nevus-like hyperplasia and melanoma in transgenic BRAFV600E mice.

BRAF, a cellular oncogene and effector of RAS-mediated signaling, is activated by mutation in approximately 60% of melanomas. Most of these mutations consist of a V600E substitution resulting in constitutive kinase activation. Mutant BRAF thus represents an important therapeutic target in melanoma. In an effort to produce a pre-clinical model of mutant BRAF function in melanoma, we have generated a mouse expressing BRAF V600E targeted to melanocytes. We show that in these transgenic mice, widespread benign melanocytic hyperplasia with histological features of nevi occurs, with biochemical evidence of senescence. Melanocytic hyperplasia progresses to overt melanoma with an incidence dependent on BRAF expression levels. Melanomas show CDKN2A loss, and genetic disruption of the CDKN2A locus greatly enhances melanoma formation, consistent with collaboration between BRAF activation and CDKN2A loss suggested from studies of human melanoma. The development of melanoma also involves activation of the Mapk and Akt signaling pathways and loss of senescence, findings that faithfully recapitulate those seen in human melanomas. This murine model of mutant BRAF-induced melanoma formation thus provides an important tool for identifying further genetic alterations that cooperates with BRAF and that may be useful in enhancing susceptibility to BRAF-targeted therapeutics in melanoma.

Cell cycle dysregulation in oral cancer.

The dysregulation of the molecular events governing cell cycle control is emerging as a central theme of oral carcinogenesis. Regulatory pathways responding to extracellular signaling or intracellular stress and DNA damage converge on the cell cycle apparatus. Abrogation of mitogenic and anti-mitogenic response regulatory proteins, such as the retinoblastoma tumor suppressor protein (pRB), cyclin D1, cyclin-dependent kinase (CDK) 6, and CDK inhibitors (p21(WAF1/CIP1), p27(KIP1), and p16(INK4a)), occur frequently in human oral cancers. Cellular responses to metabolic stress or genomic damage through p53 and related pathways that block cell cycle progression are also altered during oral carcinogenesis. In addition, new pathways and cell cycle regulatory proteins, such as p12(DOC-1), are being discovered. The multistep process of oral carcinogenesis likely involves functional alteration of cell cycle regulatory members combined with escape from cellular senescence and apoptotic signaling pathways. Detailing the molecular alterations and understanding the functional consequences of the dysregulation of the cell cycle apparatus in the malignant oral keratinocyte will uncover novel diagnostic and therapeutic approaches.

The retinoblastoma protein acts as a transcriptional coactivator required for osteogenic differentiation.

The incidence of osteosarcoma is increased 500-fold in patients who inherit mutations in the RB gene. To understand why the retinoblastoma protein (pRb) is specifically targeted in osteosarcoma, we studied its function in osteogenesis. Loss of pRb but not p107 or p130 blocks late osteoblast differentiation. pRb physically interacts with the osteoblast transcription factor, CBFA1, and associates with osteoblast-specific promoters in vivo in a CBFA1-dependent fashion. Association of pRb with CBFA1 and promoter sequences results in synergistic transactivation of an osteoblast-specific reporter. This transactivation function is lost in tumor-derived pRb mutants, underscoring a potential role in tumor suppression. Thus, pRb functions as a direct transcriptional coactivator promoting osteoblast differentiation, which may contribute to the targeting of pRb in osteosarcoma.

Requirement for p27(KIP1) in retinoblastoma protein-mediated senescence.

In vivo and in vitro evidence indicate that cells do not divide indefinitely but instead stop growing and undergo a process termed cellular proliferative senescence. Very little is known about how senescence occurs, but there are several indications that the retinoblastoma protein (pRb) is involved, the most striking being that reintroduction of RB into RB(-/-) tumor cell lines induces senescence. In investigating the mechanism by which pRb induces senescence, we have found that pRb causes a posttranscriptional accumulation of the cyclin-dependent kinase inhibitor p27(KIP1) that is accompanied by an increase in p27(KIP1) specifically bound to cyclin E and a concomitant decrease in cyclin E-associated kinase activity. In contrast, pRb-related proteins p107 and p130, which also decrease cyclin E-kinase activity, do not cause an accumulation of p27(KIP1) and induce senescence poorly. In addition, the use of pRb proteins mutated in the pocket domain demonstrates that pRb upregulation of p27(KIP1) and senescence induction do not require the interaction of pRb with E2F. Furthermore, ectopic expression of p21(CIP1) or p27(KIP1) induces senescence but not the morphology change associated with pRb-mediated senescence, uncoupling senescence from the morphological transformation. Finally, the ability of pRb to maintain cell cycle arrest and induce senescence is reversibly abrogated by ablation of p27(KIP1) expression. These findings suggest that prolonged cell cycle arrest through the persistent and specific inhibition of cdk2 activity by p27(KIP1) is critical for pRb-induced senescence.

A common polymorphism acts as an intragenic modifier of mutant p53 behaviour.

The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.

cdk6 can shorten G(1) phase dependent upon the N-terminal INK4 interaction domain.

Deregulated activity of cdk4 or cdk6 can lead to inappropriate cellular proliferation and tumorigenesis accompanied by unchecked inactivation of the retinoblastoma tumor suppressor protein. Certain tumor types preferentially activate either cdk4 or cdk6, suggesting that these kinases may not be equivalently oncogenic in all cell types. Although it is clear that cdk4 can act as an oncogene at least in part by evading inhibition by p16(INK4a), the role of cdk6 in tumorigenesis is less well understood. To investigate the consequences of aberrant expression of cdk6, the requirements for proliferation caused by cdk6 overexpression were studied. cdk6-transfected U2OS cells displayed an accelerated progression through G(1) phase that was dependent on kinase activity and that did not correlate with p27 binding. Furthermore, a mutation that prevents cdk6 interaction with INK4 proteins (cdk6R31C) was found to inactivate the proliferative effect of cdk6 and increase cytoplasmic localization, despite the fact that this mutant could phosphorylate the retinoblastoma protein in vitro. Together, these data suggest a role for the cdk6 INK4 interaction domain in the generation of functional, nuclear cdk6 complexes and demonstrate the importance of elevated cdk6 kinase activity in G(1) acceleration.

Induction of DNA synthesis and apoptosis by regulated inactivation of a temperature-sensitive retinoblastoma protein.

The retinoblastoma protein, pRb, controls entry into the S phase of the cell cycle and acts as a tumor suppressor in many tissues. Re-introduction of pRb into tumor cells lacking this protein results in growth arrest, due in part to transcriptional repression of genes required for S phase. Several studies suggest that pRb may also be involved in terminal cell cycle exit as a result of the instigation of senescence or differentiation programs. To understand better these multiple growth-inhibitory properties of pRb, a temperature-sensitive mutant of pRb has been produced. This tspRb induces G1 arrest and morphological changes efficiently at the permissive temperature of 32.5 degrees C, but is weakly functional at 37 degrees C. Consistent with this, tspRb is compromised in nuclear association and E2F regulation at the non-permissive temperature, but regains these properties at 32.5 degrees C. Serial activation and inactivation of tspRb in SAOS-2 cells does not allow proliferation, but rather leads to apoptotic cell death. Transient activation of pRb may kill tumor cells by establishing a conflict between persistent proliferation-inhibitory signals and renewed deregulation of pRb targets such as E2F, and may thus be a more potent means of eliminating these cells than through simple re-introduction of the tumor suppressor gene.

CD4+-T-cell and CD20+-B-cell changes predict rapid disease progression after simian-human immunodeficiency virus infection in macaques.

Simian-human immunodeficiency virus 89.6PD (SHIV89.6PD) was pathogenic after intrarectal inoculation of rhesus macaques. Infection was achieved with a minimum of 2,500 tissue culture infectious doses of cell-free virus stock, and there was no evidence for transient viremia in animals receiving subinfectious doses by the intrarectal route. Some animals experienced rapid progression of disease characterized by loss of greater than 90% of circulating CD4+ T cells, sustained decreases in CD20+ B cells, failure to elicit virus-binding antibodies in plasma, and high levels of antigenemia. Slower-progressing animals had moderate but varying losses of CD4+ T cells; showed increases in circulating CD20+ B cells; mounted vigorous responses to antibodies in plasma, including neutralizing antibodies; and had low or undetectable levels of antigenemia. Rapid progression led to death within 30 weeks after intrarectal inoculation. Plasma antigenemia at 2 weeks after inoculation (P < or = 0.002), B- and T-cell losses (P < or = 0.013), and failure to seroconvert (P < or = 0.005) were correlated statistically with rapid progression. Correlations were evident by 2 to 4 weeks after intrarectal SHIV inoculation, indicating that early events in the host-pathogen interaction determined the clinical outcome.

Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2'-deoxycytidine treatment induces a senescence-like state.

We have previously reported that a set of oral squamous cell carcinoma lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16ink4a, the other cell lines express undetectable levels of p16ink4a, despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16ink4A promoter and a third cell line, SCC9, has a mutation in the p16ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16ink4a. In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16ink4a persisted, even after the drug was removed and the cells expressed senescence-associated beta-galactosidase activity. Ectopic expression of p16ink4a with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype. Hence, it was possible to restore a functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16ink4a in response to treatment with a demethylating agent.

Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor.

Elevated activity of cyclin-dependent kinase 6 in human squamous cell carcinoma lines.

We have investigated the functional integrity of the retinoblastoma tumor suppressor pathway in five human squamous cell carcinoma lines. Elevated activity of cyclin-dependent kinase 6 (cdk6), a pRB kinase, was detected in all five squamous cell carcinoma lines. Overexpression of the cdk6 protein was detected in one of the five cell lines. The cdk6-specific inhibitor p18ink4C is expressed and associated with cdk6 in all five squamous cell carcinoma lines. In contrast, only very low levels of p16ink4A were detected in these cell lines. This may contribute to the elevated activity of cdk6 in these lines. Elevated activity of cdk6 may result in hyperphosphorylation of the retinoblastoma protein and, therefore, compromise its negative growth-regulatory activity.

The lymphocytosis-promoting agent pertussis toxin affects virus burden and lymphocyte distribution in the SIV-infected rhesus macaque.

Pertussis toxin from the gram-negative bacterium Bordetella pertussis is an ADP-ribosylase that modifies Gi proteins in mammalian lymphocytes and inhibits their capacity to traffic from blood into lymphoid tissues. We used this compound to induce lymphocytosis in rhesus macaques and to study its effects on SIV infection. Pertussis toxin injected at 25 micrograms/kg induced a transient lymphocytosis that peaked 3-8 days after administration and caused a rapid, transient decrease in the frequency of infectious cells in blood as judged by in vitro virus isolation assays. Lymphocyte subsets were altered during the lymphocytosis interval and sustained changes in CD8+ T cell levels were noted as long as 53 days after pertussis toxin injection. In situ hybridization studies showed that pertussis toxin altered the distribution of viral RNA in lymph nodes during the interval of lymphocytosis, and caused long-term changes with decreased virus replication in some tissue specimens.

Pertussis toxin induces lymphocytosis in rhesus macaques.

Lymph nodes and other solid tissues of the immune system are the principal sites for antigen presentation and lymphocyte activation. Lymphocytes in peripheral blood recognize the high endothelial venules within lymphoid tissues and cross from blood to tissue by the process of extravasation. Pertussis toxin is known to block extravasation and cause lymphocytosis in murine models but has not been studied extensively in nonhuman primates. We used intravenous injection of soluble pertussis toxin to induce a transient lymphocytosis in rhesus monkeys. The increase in total white blood cells was proportionally greater for lymphocytes than for polymorphonuclear cells and the CD4+ lymphocyte subpopulation increased more than the CD8+ cell population. The presence of immature polymorphonuclear cells suggested some activation of bone marrow. Clinical chemistry studies revealed an effect of pertussis toxin on liver function. Pertussis toxin is a powerful immunomodulatory agent that can disrupt and reorganize solid lymphoid tissues.

Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases.

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.

The retinoblastoma tumor suppressor protein.

Loss of the retinoblastoma protein, pRb, appears to have a role in several human tumor types. Mice lacking pRb have been produced as models of human disease, but have a different spectrum of affected tissues. Recent work shows that the tumorigenic effects of pRb may be revealed only after additional genetic alterations, such as loss of p53. New targets/effectors of pRb have been identified recently, and the system of kinases that inactivate pRb is proving to be complex.

Transcriptional regulation of neu by RB and E1A in rat-1 cells.

Functional inactivation of the tumor-suppressing retinoblastoma gene (Rb) is involved in the etiology of many types of human cancers, including hereditary retinoblastomas. The neu gene is a dominant transforming oncogene, and we previously found that the Rb-encoded protein (RB) suppresses neu-induced transformation in NIH3T3 cells by repressing transcription of the neu oncogene. We report here that RB was unable to repress neu oncogene transcription in Rat-1 cells but could functionally antagonize transcriptional repression of neu by the adenovirus E1A. Mutant forms of RB that have mutations in either the E1A-binding or carboxy-terminal regions had less or no antagonizing effects on E1A-mediated repression of neu in Rat-1 cells. Results of focus-formation assays showed that the transformation activity of the neu oncogene in Rat-1 cells could be regulated by E1A and RB in accordance with their transcriptional regulation activities. The data demonstrate that RB can regulate transcription of neu in a negative or positive manner depending on the cell type. Carboxy terminus of RB as well as the E1A-binding region can mediate transcriptional regulation. Based on these results, we propose a model for the complex transcriptional regulation of neu by RB and E1A.

Tumor suppressor genes.

The mutation of tumor suppressor genes is thought to contribute to tumor growth by inactivating proteins that normally act to limit cell proliferation. Several tumor suppressor proteins have been identified in recent years, but only two of them, p53 and pRb, are understood in detail. In the past year, a role has become apparent for both of these proteins in transcription and phosphorylation events required for passage of a cell from G1 to S phase. The pRb protein appears to prevent the function of transcription factors and other proteins needed for S phase until its inactivation by cyclin-dependent kinases in late G1. Induction of p53 by DNA damage may act to cause cell cycle arrest or cell death by altering the transcription program of damaged cells. A detailed molecular understanding of these growth regulators is now emerging, and is the subject of this review.