Identifying lifestyle factors associated to co-morbidity of obesity and psychiatric disorders, a pilot study.

Obesity and psychiatric disorders are linked through a bidirectional association. Obesity rates have tripled globally in the past decades, and it is predicted that by 2025, one billion people will be affected by obesity, often with a co-morbidity such as depression. While this co-morbidity seems to be a global health issue, lifestyle factors associated to it differ between countries and are often attributed to more than one factor. Prior obesity studies were performed in Western populations; this is the first study that investigates lifestyle factors relating to obesity and mental health of the diverse population in Qatar, a country that has witnessed tremendous lifestyle change in a short time. In this pilot study, we surveyed 379 respondents to assess and compare the lifestyles of Qatar residents to the global population. However due to the high proportion of responses from the United Kingdom (UK) residents, we have made comparisons between Qatar residents and UK residents. We used chi-square analysis, spearman rank correlation and logistic regression to compare the lifestyle factors of individuals suffering from both increased BMI and mental health conditions. The types of food consumed, stress, exercise frequency and duration, alcohol and tobacco consumption, and sleep duration, were explored and results argue that different lifestyle factors can contribute to the same health condition, suggesting different mechanisms involved. We found that both groups reported similar sleep durations (p = 0.800), but that perception of sleep (p = 0.011), consumption of alcohol (p = 0.001), consumption of takeaway food (p = 0.007), and physical activity significantly varied between the groups (p = 0.0001). The study examined the predictors of comorbidity in Qatar as well as UK populations using multivariate logistic regression analysis. The result of the study showed no statistical association between comorbidity and the predictors drinking habit, smoking, physical activity, vegetable consumption, eat outs, and sleep perception for the Qatar population, and for the combined population. This study, however showed a significant association (p = 0.033) between sleep perception and comorbidity for the UK population. We conclude that further analysis is needed to understand the relationship between specific lifestyle factors and multimorbidity in each country.

Nanoparticle-mediated cancer cell therapy: basic science to clinical applications.

Every sixth person in the world dies due to cancer, making it the second leading severe cause of death after cardiovascular diseases. According to WHO, cancer claimed nearly 10 million deaths in 2020. The most common types of cancers reported have been breast (lung, colon and rectum, prostate cases), skin (non-melanoma) and stomach. In addition to surgery, the most widely used traditional types of anti-cancer treatment are radio- and chemotherapy. However, these do not distinguish between normal and malignant cells. Additional treatment methods have evolved over time for early detection and targeted therapy of cancer. However, each method has its limitations and the associated treatment costs are quite high with adverse effects on the quality of life of patients. Use of individual atoms or a cluster of atoms (nanoparticles) can cause a paradigm shift by virtue of providing point of sight sensing and diagnosis of cancer. Nanoparticles (1-100 nm in size) are 1000 times smaller in size than the human cell and endowed with safer relocation capability to attack mechanically and chemically at a precise location which is one avenue that can be used to destroy cancer cells precisely. This review summarises the extant understanding and the work done in this area to pave the way for physicians to accelerate the use of hybrid mode of treatments by leveraging the use of various nanoparticles.

Highlights on selected growth factors and their receptors as promising anticancer drug targets.

Growth factor receptors (GFRs) and receptor tyrosine kinases (RTK) are groups of proteins mediating a plethora of physiological processes, including cell growth, proliferation, survival, differentiation and migration. Under certain circumstances, expression of GFRs and subsequently their downstream kinase signaling are deregulated by genetic, epigenetic, and somatic changes leading to uncontrolled cell division in many human diseases, most notably cancer. Cancer cells rely on growth factors to sustain the increasing need to cell division and metabolic reprogramming through cancer-associated activating mutations of their receptors (i.e., GFRs). In this review, we highlight the recent advances of selected GFRs and their ligands (growth factors) in cancer with emphasis on structural and functional differences. We also interrogate how overexpression and/or hyperactivation of GFRs contribute to cancer initiation, development, progression, and resistance to conventional chemo- and radiotherapies. Novel approaches are being developed as anticancer agents to target growth factor receptors and their signaling pathways in different cancers. Here, we illustrate how the current knowledge of GFRs biology, and their ligands lead to development of targeted therapies to inhibit and/or block the activity of growth factors, GFRs and downstream kinases to treat diseases such as cancer.

Exopolysaccharide-peptide complex from oyster mushroom (Pleurotus ostreatus) protects against hepatotoxicity in rats.

Liver damage involves oxidative stress and a progression from chronic hepatitis to hepatocellular carcinoma (HCC). The increased incidence of liver disease in Egypt and other countries in the last decade, coupled with poor prognosis, justify the critical need to introduce alternative chemopreventive agents that may protect against liver damage. The aim of this study was to evaluate the efficacy of exopolysaccharide-peptide (PSP) complex extracted from Pleurotus ostreatus as a hepatoprotective agent against diethylnitrosamine (DEN)/carbon tetrachloride (CCL4)-induced hepatocellular damage in rats. The levels of liver injury markers (ALT, AST and ALP) were substantially increased following DEN/CCl4 treatment. DEN/CCl4 - induced oxidative stress was confirmed by elevated levels of lipid peroxidation and decreased levels of superoxide dismutase, glutathione-S-transferase, and reduced glutathione. PSP reversed these alterations in the liver and serum, and provided protection evidenced by reversal of histopathological changes in the liver. The present study demonstrated that PSP extract from P. ostreatus exhibited hepatoprotective and antioxidant effects against DEN/CCl4-induced hepatocellular damage in rats. Given the high prevalence of HCV-related liver damage in Egypt, our results suggest further clinical evaluation of P. ostreatus extracts and their potential hepatoprotective effects in patients with liver disease.

Clinical resistance associated with a novel MAP2K1 mutation in a patient with Langerhans cell histiocytosis.

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment-refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

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.

Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells.

BACKGROUND: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. METHODS: Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. RESULTS: Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the ß5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. CONCLUSIONS: We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.

Downregulation of IGF-1 receptor occurs after hepatic linage commitment during hepatocyte differentiation from human embryonic stem cells.

The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyte differentiation. Human hepatocyte cancer cells and stem cells are known to express IGF-1R whereas normal hepatocytes do not. In the present study we optimized a differentiation protocol and verified the different stages by established markers. The expression levels of IGF-1R and major downstream signaling proteins during differentiation from human embryonic stem cells (hESC) to mature hepatocytes were investigated. We could only demonstrate a minor decrease in IGF-1R expression during endodermal differentiation compared to hESC, but declined substantially (>50%) after hepatic lineage commitment during the hepatocyte specification and maturation stages. This downregulation was paralleled by an upregulation of ERK 1/2, AKT and insulin substrate-1. Neither inhibition nor activation of IGF-1R had any essential effect on endoderm differentiation of human embryonic stem cells. Therefore, our data suggest that IGF-1R downregulation may have a regulatory impact after initiation of hepatic lineage commitment.

Targeting cell cycle regulators in hematologic malignancies.

Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via insulin-like growth factor-1 receptor-independent mechanism.

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.

CDK2 knockdown enhances head and neck cancer cell radiosensitivity.

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.

ß-Glucans and their applications in cancer therapy: focus on human studies.

ß-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 ß-glucans are glucose polymers linked together by a (ß 1-3) linear ß-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 ß- glucans were performed, but their anticancer potential was demonstrated mainly through studies using extracts from mushrooms, yeast or other sources which contain ß-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. ß-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 ß- glucans in cancer treatment and prevention. The potential direct effects of ß-glucans on cancer cells are also described.

Serum IGFBP-3 is a more effective predictor than IGF-1 and IGF-2 for the development of hepatocellular carcinoma in patients with chronic HCV infection.

Hepatocellular carcinoma (HCC) contributes to 14.8% of all cancer mortality in Egypt, which has a high prevalence of hepatitis C virus (HCV). We have previously shown alterations in the insulin-like growth factor-1 (IGF-1) receptor signalling pathway during experimental hepatocarcinogenesis. The aim of this study was to determine whether serum levels of IGF-1, IGF-2 and IGFBP-3 can be used to discriminate between HCC and the stages of hepatic dysfunction in patients with liver cirrhosis assessed by the Child-Pugh (CP) score, and to correlate these levels with HCC stages. We recruited 241 subjects to the present study; 79 with liver cirrhosis, 62 with HCV-induced HCC and 100 age-matched controls. Results showed that serum levels of IGF-1, IGF-2 and IGFBP-3 were reduced significantly in cirrhosis and HCC patients in comparison to the controls, and that this reduction negatively correlated with the CP scores. However, only IGFBP-3 levels showed significant negative correlation with α-fetoprotein levels. The reduction in IGF-1 and IGFBP-3 but not IGF-2 levels was significant in HCC in comparison to patients with cirrhosis. None of the parameters significantly correlated with the HCC stage. IGFBP-3 levels discriminated between cirrhosis and HCC at a sensitivity of 87%, a specificity of 80% and a cut-off value of <682.6 ng/ml. In conclusion, although our results showed that serum IGF-1, IGF-2 and IGFBP-3 are reduced with the progression of hepatic dysfunction, only IGFBP-3 may be considered as the most promising serological marker for the prediction of the development of HCC in the chronic HCV patients with liver cirrhosis.

Schizophyllan inhibits the development of mammary and hepatic carcinomas induced by 7,12 dimethylbenz(α)anthracene and decreases cell proliferation: comparison with tamoxifen.

BACKGROUND: 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 ß-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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Multiple antitumor effects of picropodophyllin in colon carcinoma cell lines: clinical implications.

Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1R-mediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 µM PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease.

Upregulation of the insulin receptor and type I insulin-like growth factor receptor are early events in hepatocarcinogenesis.

The molecular mechanisms underlying the development of hepatocellular carcinoma (HCC) are not yet fully understood. Preneoplastic foci of altered hepatocytes regularly precede HCC in various species. The predominant earliest type of foci of altered hepatocytes, the glycogen storage focus (GSF), shows an excess of glycogen (glycogenosis) in the cytoplasm. During progression from GSF to HCC, the stored glycogen is gradually reduced, resulting in complete loss in basophilic HCC. We have previously shown that in N-nitrosomorpholine-induced hepatocarcinogenesis, insulin receptor substrate (IRS-1) is strongly expressed in GSF and reduced during progression to HCC, thus correlating with the glycogen content. In the present study, we observed increased levels of insulin receptor, IGF-I receptor (IGF-IR), IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 in GSF, following the same pattern of expression as IRS-1. We conclude that the abundance of IRS-1, IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 coincides with a concerted upregulation of both IR and IGF-IR induced by the hepatocarcinogen. Our data suggest that in early hepatocellular preneoplasia, the upregulation of IR elicits glycogenosis through IRS-1 and/or IRS-2, whereas the increased level of the IGF-IR may lead to the increased cell proliferation previously reported in GSF. Therefore, the concerted upregulation of both IR and IGF-IR may represent initial events in hepatocarcinogenesis.

Cdk2 and Cdk4 activities are dispensable for tumorigenesis caused by the loss of p53.

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.

PRKAR1A inactivation leads to increased proliferation and decreased apoptosis in human B lymphocytes.

The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIalpha regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIalpha levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur.

Dependence of cisplatin-induced cell death in vitro and in vivo on cyclin-dependent kinase 2.

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.

Mouse models of cell cycle regulators: new paradigms.

In yeast, a single cyclin-dependent kinase (Cdk) is able to regulate diverse cell cycle transitions (S and M phases) by associating with multiple stage-specific cyclins. The evolution of multicellular organisms brought additional layers of cell cycle regulation in the form of numerous Cdks, cyclins and Cdk inhibitors to reflect the higher levels of organismal complexity. Our current knowledge about the mammalian cell cycle emerged from early experiments using human and rodent cell lines, from which we built the current textbook model of cell cycle regulation. In this model, the functions of different cyclin/Cdk complexes were thought to be specific for each cell cycle phase. In the last decade, studies using genetically engineered mice in which cell cycle regulators were targeted revealed many surprises. We discovered the in vivo functions of cell cycle proteins within the context of a living animal and whether they are essential for animal development. In this review, we discuss first the textbook model of cell cycle regulation, followed by a global overview of data obtained from different mouse models. We describe the similarities and differences between the phenotypes of different mouse models including embryonic lethality, sterility, hematopoietic, pancreatic, and placental defects. We also describe the role of key cell cycle regulators in the development of tumors in mice, and the implications of these data for human cancer. Furthermore, animal models in which two or more genes are ablated revealed which cell cycle regulators interact genetically and functionally complement each other. We discuss for example the interaction of cyclin D1 and p27 and the compensation of Cdk2 by Cdc2. We also focus on new functions discovered for certain cell cycle regulators such as the regulation of S phase by Cdc2 and the role of p27 in regulating cell migration. Finally, we conclude the chapter by discussing the limitations of animal models and to what extent can the recent findings be reconciled with the past work to come up with a new model for cell cycle regulation with high levels of redundancy among the molecular players.