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We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP.

PURPOSE: Cyclin-dependent kinase 2 (CDK2) is critically involved in cell cycling and has been proposed as a potential cancer target. It remains largely elusive whether CDK2 targeting alters the tumor cell radiosensitivity. MATERIALS AND METHODS: CDK2(-/-) and wild type (WT) mouse embryonic fibroblasts (MEF) as well as six human head and neck squamous cell carcinoma (HNSCC) cell lines (SAS, FaDu, Cal-33, HSC-4, UTSCC-5, UTSCC-8) were used. Upon CDK2 knockdown using small interfering technology, colony formation, DNA double-strand breaks (DSB), cell cycle distribution and expression and phosphorylation of major proteins regulating cell cycle and DNA damage repair were examined. RESULTS: CDK2(-/-) MEF and CDK2 HNSCC knockdown cell cultures were more radiosensitive than the corresponding controls. Repair of DSB was attenuated under CDK2 knockout or knockdown. In contrast to data in MEF, combined CDK2 knockdown with irradiation showed no cell cycling alterations in SAS and FaDu cultures. Importantly, CDK2 knockdown failed to radiosensitize SAS and FaDu when cultured in a more physiological three-dimensional (3D) extracellular matrix environment. CONCLUSIONS: Our findings suggest that targeting of CDK2 radiosensitizes HNSCC cells growing as monolayer. Additional studies performed under more physiological conditions are warranted to clarify the potential of CDK2 as target in radiotherapy.

Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compounds have been isolated from mushrooms. The aim of the present work was to study the anticancer effects of schizophyllan (SCH), a β-d-glucan extracted from the mushroom Schizophyllum commune alone or in combination with tamoxifen (TAM) on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinomas in mice. We isolated SCH from S. commune. Female mice received DMBA, SCH, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH or vehicles. We studied mice survival, tumour incidence, histopathology, oestrogen receptor (ER) expression, cell proliferation by immunohistochemical detection of proliferating cell nuclear antigen (PCNA), apoptosis by TUNEL assay, as well as caspase-3 expression. DMBA treatment resulted in mammary and hepatocellular carcinomas (HCC). Both SCH and TAM reduced the incidence of DMBA-induced mammary tumours by 85 and 75 %, respectively, and equally decreased the PCNA labelling index relative to DMBA. TAM treatment increased the incidence of- and PCNA index in HCCs relative to DMBA, while SCH suppressed these effects. TAM was more effective than SCH in the induction of apoptosis in both mammary and hepatic carcinomas. Caspase-3 levels correlated with the apoptotic index in most experimental groups. Only one dose of SCH had similar therapeutic effects against DMBA-induced mammary carcinomas as 4 weeks of TAM treatment. This coupled with the ability of SCH to suppress hepatic lesions associated with TAM treatment provides the rationale for further investigating the combined therapeutic effects of TAM+SCH in preclinical models of ER-positive breast cancer, as well as in liver cancer.

The loss of p53 induces spontaneous tumors in mice, and p53 mutations are found in approximately 50% of human tumors. These tumors are generally caused by a number of events, including genomic instability, checkpoint defects, mitotic defects, deregulation of transcriptional targets, impaired apoptosis, and G(1) deregulation or a combination of these effects. In order to determine the role of proteins involved in G(1) control in tumorigenesis, we focused on Cdk2 and Cdk4, two cyclin-dependent kinases that in association with cyclin E and cyclin D promote the G(1)/S phase transition. We analyzed the consequence of loss of Cdk2 in p53-null animals by generating Cdk2(-/-) p53(-/-) mice. These mice are viable and developed spontaneous tumors, predominantly lymphoblastic lymphomas, similar to p53(-/-) mice. In contrast, the genotypes Cdk4(-/-) p53(-/-) were mostly lethal, with few exceptions, and Cdk2(-/-) Cdk4(-/-) p53(-/-) mice die during embryogenesis at embryonic day 13.5. To study the oncogenic potential, we generated mouse embryonic fibroblasts (MEFs) and found that p53(-/-), Cdk2(-/-) p53(-/-), Cdk4(-/-) p53(-/-), and Cdk2(-/-) Cdk4(-/-) p53(-/-) MEFs grew at similar rates without entering senescence. Ras-transformed MEFs of these genotypes were able to form colonies in vitro and induce tumors in nude mice. Our results suggest that tumorigenicity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates considering the use of broad-spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy.

Depending upon the cellular and physiologic context, the overexpression of the MYC proto-oncogene results in rapid cell growth, proliferation, induction of apoptosis and/or proliferative arrest. What determines the precise consequences upon MYC activation is not clear. We have found that cyclin-dependent kinase 2 (CDK2) is required by MYC to induce apoptosis. MYC-induced apoptosis was suppressed in mouse embryonic fibroblasts (MEF) knocked out for Cdk2 or normal human fibroblasts (NHF) upon expression of the CDK2 inhibitor p27 or treated with RNAi directed at CDK2. Knockout of Cdk2 did not prevent MYC from inducing p53 and Bim. The inhibition of CDK2 did not prevent apoptosis induced by the DNA damaging agent etoposide. Our results surprisingly suggest that CDK2 defines whether MYC induction causes apoptosis.

Interleukin (IL)-7 is required for survival and homeostatic proliferation of T lymphocytes. The survival effect of IL-7 is primarily through regulation of Bcl-2 family members; however, the proliferative mechanism is unclear. It has not been determined whether the IL-7 receptor actually delivers a proliferative signal or whether, by promoting survival, proliferation results from signals other than the IL-7 receptor. We show that in an IL-7–dependent T cell line, cells protected from apoptosis nevertheless underwent cell cycle arrest after IL-7 withdrawal. This arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 through a posttranslational mechanism. Overexpression of p27Kip1 induced G1 arrest in the presence of IL-7, whereas knockdown of p27Kip1 by small interfering RNA promoted S phase entry after IL-7 withdrawal. CD4 or CD8 T cells transferred into IL-7–deficient hosts underwent G1 arrest, whereas 27Kip1-deficient T cells underwent proliferation. We observed that IL-7 withdrawal activated protein kinase C (PKC)θ and that inhibition of PKCθ with a pharmacological inhibitor completely blocked the rise of p27Kip1 and rescued cells from G1 arrest. The conventional pathway to breakdown of p27Kip1 is mediated by S phase kinase-associated protein 2; however, our evidence suggests that PKCθ acts via a distinct, unknown pathway inducing G1 arrest after IL-7 withdrawal from T cells. Hence, IL-7 maintains T cell proliferation through a novel pathway of p27Kip1 regulation.

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.

It has long been believed that Cdk2 and its activator cyclin E play essential roles in the progression of the mitotic cell cycle. However, recent studies using knockout mouse models revealed that neither Cdk2 nor cyclin E are essential in vivo. The purpose of this Perspective is to compare both Cdk2 and cyclin E knockout mice models and to discuss potential mechanisms driving the cell cycle in the absence of Cdk2 or cyclin E. Particular emphasis is placed on possible non-catalytic roles of cyclin E, the expression and activity of the second cyclin binding partner of Cdk2, cyclin A, as well as on the expression and degradation of the Cdk2 inhibitor p27Kip1 in the absence of Cdk2.

β-glucans belong to a group of polysaccharides located in the cell wall of bacteria, fungi including mushrooms, as well as cereals such as barley and oats. All β-glucans are glucose polymers linked together by a (β 1-3) linear β-glycosidic chain core and they differ by their length and branching structures. They are considered biological response modifiers with immunomodulatory and health beneficial effects including anticancer properties. Few studies using purified β- glucans were performed, but their anticancer potential was demonstrated mainly through studies using extracts from mushrooms, yeast or other sources which contain β-glucan as a key component. Their anticancer effects were demonstrated mainly in in vitro and in vivo experimental systems but fewer studies from human populations are available. β-glucans have been used as adjuvant therapy in clinical trials, mainly in the Far East, with a positive effect on patients'survival and quality of life. The mechanism of action is suggested to be through its stimulation of the immune system. This review focuses on human studies; clinical trials and epidemiological data assessing the efficacy and safety of mushroom-derived β- glucans in cancer treatment and prevention. The potential direct effects of β-glucans on cancer cells are also described.

Cisplatin is one of the most effective chemotherapeutics, but its usefulness is limited by its toxicity to normal tissues, including cells of the kidney proximal tubule. The purpose of these studies was to determine the mechanism of cisplatin cytotoxicity. It was shown in vivo that cisplatin administration induces upregulation of the gene for the p21 cyclin-dependent kinase (cdk) inhibitor in kidney cells. This protein is a positive effector on the fate of cisplatin-exposed renal tubule cells in vivo and in vitro; adenoviral transduction of p21 completely protected proximal tubule cells from cisplatin toxicity. Herein is reported that cdk2 inhibitory drugs protect kidney cells in vivo and in vitro, that transduction of kidney cells in vitro with dominant-negative cdk2 also protected, and that cdk2 knockout cells were resistant to cisplatin. The cdk2 knockout cells regained cisplatin sensitivity after transduction with wild-type cdk2. It is concluded that cisplatin cytotoxicity depends on cdk2 activation and that the mechanism of p21 protection is by direct inhibition of cdk2. This demonstrated the involvement of a protein that previously was associated with cell-cycle progression with pathways of apoptosis. It also was demonstrated that this pathway of cisplatin-induced cell death can be interceded in vivo to prevent nephrotoxicity.

It has been long believed that the cyclin-dependent kinase 2 (Cdk2) binds to cyclin E or cyclin A and exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a major role in mitosis. We now provide evidence that Cdc2 binds to cyclin E (in addition to cyclin A and B) and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2 can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle model and how results from knockout mice provide new evidence that refute this model. We focus on the roles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cell cycle regulation that accommodates these novel findings.

Protein phosphatase inhibitor-1 plays an important role in the regulation of glycogen metabolism through inhibition of protein phosphatase-1 activity, and it has been implicated in the regulation of cell growth. Using real-time quantitative RT-PCR, we studied the mRNA expression of inhibitor-1 in hepatocellular carcinomas induced in rats by oral administration of N-nitrosomorpholine, and in a non-tumorigenic liver cell line (C 1 I), that stores glycogen in excess during early passages. In late passages, glycogen is gradually lost concomitant with cell transformation. Our in vitro model included a tumorigenic subline of C 1 I cells that was obtained by chemically-induced neoplastic transformation using N-methyl-N'-nitro-N-nitrosoguanidine (C 1 I ct ), and does not store glycogen, as well as Morris hepatoma 3924A (MH3924A) cells. We found that in hepatocellular carcinomas, in the late glycogen-poor passages (C 1 I late ), and in the tumorigenic subline (C 1 I et ) of C 1 I cells, and in MH3924A cells the mRNA expression of inhibitor-1 is significantly increased. This increase in expression varied from 15 to 290-fold of that observed in normal liver. In contrast, in the early glycogen-storing passage of C 1 I cells (C 1 I early ) the level of inhibitor-1 mRNA was found to be slightly less than that of normal liver. Inhibitor-1 mRNA levels correlated with the degree of differentiation of HCCs. These results indicate that the expression of inhibitor-1 mRNA is tightly linked to tumor progression and to the process of liver cell transformation in vitro and is inversely correlated with the glycogen content of the cell.

Chloramphenicol has been associated with the development of aplastic anaemia. As it is still widely used in Egypt, we studied its effect on 100 Egyptian toads [Bufo regularis] given a dose of chloramphenicol of 5 mg/40 g body weight for 12 weeks. We found it induced numerous, severe ultrastructural changes in almost all types of leukocytes. These changes were similar to those induced by the chemical carcinogen 7, 12-dimethylbenz[a]anthracene in 100 toads used as the carcinogen control group, and similar to those in leukocytes reported in humans with leukaemia. We recommend regulations be applied on the use of this antibiotic in countries where it is still widely used.