Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer.

Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factor-α (TNF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.

Mesalazine and thymoquinone attenuate intestinal tumour development in Msh2(loxP/loxP) Villin-Cre mice.

OBJECTIVE: Lynch syndrome is caused by germline mutations in DNA mismatch repair genes leading to microsatellite instability (MSI) and colorectal cancer. Mesalazine, commonly used for the treatment of UC, reduces MSI in vitro. Here, we tested natural compounds for such activity and applied mesalazine and thymoquinone in a Msh2(loxP/loxP) Villin-Cre mouse model for Lynch syndrome. DESIGN: Flow cytometry was used for quantitation of mutation rates at a CA13 microsatellite in human colon cancer (HCT116) cells that had been stably transfected with pIREShyg2-enhanced green fluorescent protein/CA13, a reporter for frameshift mutations. Mice were treated for 43 weeks with mesalazine, thymoquinone or control chow. Intestines were analysed for tumour incidence, tumour multiplicity and size. MSI testing was performed from microdissected normal intestinal or tumour tissue, compared with mouse tails and quantified by the number of mutations per marker (NMPM). RESULTS: Besides mesalazine, thymoquinone significantly improved replication fidelity at 1.25 and 2.5 µM in HCT116 cells. In Msh2(loxP/loxP) Villin-Cre mice, tumour incidence was reduced by mesalazine from 94% to 69% (p=0.04) and to 56% (p=0.003) by thymoquinone. The mean number of tumours was reduced from 3.1 to 1.4 by mesalazine (p=0.004) and to 1.1 by thymoquinone (p<0.001). Interestingly, MSI was reduced in normal intestinal tissue from 1.5 to 1.2 NMPM (p=0.006) and to 1.1 NMPM (p=0.01) by mesalazine and thymoquinone, respectively. Thymoquinone, but not mesalazine, reduced MSI in tumours. CONCLUSIONS: Mesalazine and thymoquinone reduce tumour incidence and multiplicity in Msh2(loxP/loxP) Villin-Cre mice by reduction of MSI independent of a functional mismatch repair system. Both substances are candidate compounds for chemoprevention in Lynch syndrome mutation carriers.

MSH3-deficiency initiates EMAST without oncogenic transformation of human colon epithelial cells.

BACKGROUND/AIM: Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. METHODS: HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay. RESULTS: Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10(-4)) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. CONCLUSIONS: MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.

The use of molecular markers in the diagnosis of colorectal cancer screening.

BACKGROUND: Colorectal cancer (CRC) is among the most prevalent cancers. Despite remarkable advances in detection and treatment of the illness, more than a third of patients diagnosed with CRC ultimately succumb to the neoplastic lesions, mostly due to metastases. Chromosomal instability, DNA mismatch repair defects, epigenetic silencing due to aberrant methylation of promoters, defects in base excision repair and activation of oncogenic pathways are the prerequisites for CRC development. Early detection of CRC is of paramount importance and the key to ultimately curing the vast majority of patients. METHODS: Additionally to testing for fecal occult blood, assays for the detection of CRC-specific mutations and aberrant promoter methylation of fecal DNA and of DNA isolated from blood have been established. Moreover, assays for profiling DNA, RNA and proteins in plasma to detect CRC in early stages have been the focus of intense research. RESULTS: The improved and newly developed assays described in this article show the potential for strongly improved sensitivity at high specificity. Comparison of various assays shows that the accuracy of the tests strongly depends on the experimental setup and the samples used (e.g. blood vs. stool DNA, early vs. late cancer stage). CONCLUSION: The broad implementation of screens for CRC with high sensitivity and specificity, as developed in recent years, should strongly increase the early detection of neoplastic transformations and thereby, the survival rate of cancer patients.

Oncogenes do not Fully Override Cell-intrinsic Traits: Pronounced Impact of the Cellular Programme.

Overexpression of p53 tumor suppressor protein in malignant cells induces cell cycle arrest, or alternatively, apoptosis thereby indicating that additional factors may contribute to the p53-mediated outcome. Comparison of the experimental protocols revealed that the construct encoding wild-type (wt) p53 was expressed in cells of different origin. Therefore, we decided to determine whether the intrinsic cellular program of primary cells of the same genetic background could have any effect on the oncogenic potential of mutated c-Ha-RAS and TP53. Primary rat cells (RECs) isolated from rat embryos of different age: at 13.5 gd (y) and 15.5 gd (o), were used for transfection. Immortalized rat cell clones overexpressing temperature-sensitive (ts) p53(135val) mutant and transformed cell clones after co-transfection with oncogenic c-Ha-Ras, were generated. The ts p53(135Val) mutant, switching between wt and mutant conformation, offers the possibility to study the role of p53 in cell cycle control in a model of malignant transformation in cells with the same genetic background. Surprisingly, the kinetics of cell proliferation at non-permissive temperature and that of cell cycle arrest at 32°C strongly differed between cell clones established from yRECs and oRECs. Furthermore, the kinetics of the re-enter of G1-arrested cells in the active cell cycle strongly differed between distinct cell clones. Finally, the susceptibility of immortalized and transformed cells to the pharmacological inhibitors of cyclin-dependent kinases (CDKs) considerably differed. Our results clearly show that overexpression of genes such as mutated TP53 and oncogenic c-Ha-RAS is not able to fully override the intrinsic cellular programme.

p53-mediated regulation of cell cycle progression: pronounced impact of cellular microenvironment.

Data on the biological effects of some overexpressed oncogenes and their cooperation with cellular factors are, at least partially, contradictory. There are reports on the strong pro-apoptotic action of temperature-sensitive (ts) p53(135val) in transformed cells at permissive temperature. However, in our experience very high levels of p53(135val) induce in transformed rat cells at permissive temperature cell cycle arrest but not apoptosis. Comparison of the experimental protocols reveals that cells used for transfection strongly differ. Therefore, we decided to explore the impact of primary cells used for generation of cell clones on the biological effects evoked by p53 and c-Ha-Ras. In the present study, we used primary rat cells (RECs) isolated from rat embryos of different age: at 13.5 gd (y) and 15.5 gd (o). We immortalized rat cells using ts p53(135val) mutant and additionally generated transformed cells after co-transfection with oncogenic Ras. The RECs were transfected with a constitutively activated Ha-Ras protein, a mutation that is found in a wide variety of human tumors. The ts p53(135Val) mutant, switching between wild-type (wt) and mutant conformation, offers the possibility to study the escape from p53-mediated cell cycle control in a model of malignant transformation in cells with the same genetic background. Surprisingly, the kinetics of cell proliferation at non-permissive temperature and that of cell cycle arrest at 32 degrees C strongly differed between cell clones established from yRECs and oRECs, thereby indicating that overexpression of genes such as ts p53(135Val) mutant and oncogenic-Ha-Ras does not fully override the intrinsic cellular program.

A combined treatment of HeLa cells with the farnesyl protein transferase inhibitor L-744,832 and cisplatin significantly increases the therapeutic effect as compared to cisplatin monotherapy.

Activating mutations of Ras that frequently occur during malignant transformation, enhance growth-promoting signal transduction, allowing cells to bypass stringent control of cell cycle progression, thereby rendering them highly proliferative. Abundantly expressed c-Ha-ras protein in human cervical HeLa cells is farnesylated and attached to the plasma membrane, inducing enhanced signal transduction. Exposure of HeLa cells to cisplatin very efficiently inhibits cell proliferation and induces apoptosis. Unfortunately, high doses of cisplatin are strongly cytotoxic, therefore, an alternative therapeutic strategy allowing dose reduction of cisplatin by inhibition of farnesylation could increase the curative effects of cisplatin, thereby benefiting cancer patients. We used two inhibitors of farnesyl protein transferase (FPTase), FTI, and L-744,832, to sensitize HeLa cells to the action of cisplatin. The combined administration of cisplatin and inhibitors of FPTase increased the cytostatic potency of cisplatin. L-744,832 exhibited a stronger synergistic effect in combination with cisplatin than FTI. Moreover, the efficiency of the combined therapy strongly depended on the treatment regimen: The highest efficiency was achieved after combined treatment for 24 h and post-incubation with an inhibitor of FPTase for 48 h. Following this optimized treatment, apoptosis was induced in approximately 50% of HeLa cells treated with 1 microM cisplatin, representing approximately a threefold increase as compared to cisplatin monotherapy. Combined treatment of HeLa cells with cisplatin and inhibitors of FPTase significantly increases the efficacy of the therapy and allows to reduce the dose of cisplatin. Importantly, best therapeutic effects can be achieved by post-treatment with inhibitors of FPTase.

Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context.

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.

Effect of Src kinase inhibition on metastasis and tumor angiogenesis in human pancreatic cancer.

Tumor angiogenesis is a process that requires migration, proliferation, and differentiation of endothelial cells. We hypothesized that decrease in pancreatic tumor growth due to inhibition of Src activity is associated with the inability of Src kinase to trigger a network of such signaling processes, which finally leads to endothelial cell death and angiogenesis-restricted tumor dormancy. The therapeutic efficacy of Src kinase inhibitor AZM475271 was tested in nude mice orthotopically xenografted with L3.6pl pancreatic carcinoma cells. No liver metastases and peritoneal carcinosis were detected and a significant effect on the average pancreatic tumor burden was observed following treatment with AZM475271, which in turn correlated with a decrease in cell proliferation and an increase in apoptotic endothelial cells. AZM475271 was shown to significantly inhibit migration of human umbilical vein endothelial cells in an in vitro Boyden Chamber cell migration assay. In a rat aortic ring assay we could demonstrate as well inhibition of endothelial cell migration and sprouting following therapy with Src kinase inhibitor at similar doses. The most conclusive anti-angiogenic activity of AZM475271 was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor-induced neovascularization in response to systemic administration of AZM475271. Furthermore, we could show reduced proliferation of HUVECs determined with the TACS MTT Cell Viability Assay Kit. The blockade of Src kinase significantly reduced the level of VEGF in L3.6pl medium, the effect which was found also in the cell culture supernate from HUVECs. Inhibition of Src kinase by AZM475271 also showed prevention of survival signaling from VEGF and EGF receptors. Treatment with AZM475271 resulted in VEGF - dependent inhibition of tyrosine phosphorylation of FAK. HUVECs were also examined using propidium iodide staining for cell cycle analysis by FACS. Inhibition of Src kinase promoted HUVEC apoptosis in a dose-dependent manner. Taken together, our results suggest that the Src kinase inhibitor AZM475271, in addition to its effects on tumor cells, suppresses tumor growth and metastasis in vitro and in vivo potentially also by anti-angiogenic mechanisms.

Cellular and organismal ageing: Role of the p53 tumor suppressor protein in the induction of transient and terminal senescence.

In recent years, an impact of the p53 tumor suppressor protein in the processes of cellular and organismal ageing became evident. First hints were found in model organisms like Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster where a clear connection between ageing phenotypes and pathways that are regulated by p53, were found. Interestingly, pathways that are central to the ageing process are usually also involved in energy metabolism and are highly conserved throughout evolution. This also supports the long known empiric finding that caloric restriction has a positive impact on the life span of a wide variety of organisms. Within the last years, on the molecular level, an involvement of the insulin-like growth factor and of the histone deacetylase SRIT1 could be shown. Insight on the impact of p53 on ageing at the organismal level came from mice expressing aberrant forms of the p53 protein. Obviously, the balance of the full length p53 protein and of the shorter p44/DeltaNp53 isomer bear a strong impact on ageing. The shorter isoform regulates full length p53 and in cases where there is too much of the longer isoform, this leads to elevated apoptosis resulting in decreased tumor incidence but also in premature ageing due to exhaustion of the renewal potential. Therefore, modulating the expression of the truncated p53 isoform accordingly, might lead to increased health-span and elevated life-span.

Inhibition of farnesyl protein transferase sensitizes human MCF-7 breast cancer cells to roscovitine-mediated cell cycle arrest.

We reported recently that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrests human MCF-7 breast cancer cells in G(2) phase of the cell cycle, and concomitantly induces apoptosis. Human MCF-7 breast cancer cells are known to express elevated levels of c-Ha-Ras protein. To achieve full biological activity, de novo synthesized c-Ha-Ras protein has to be farnesylated and after further processing it needs to be attached to the plasma membrane. Therefore, we decided to prove whether prevention of protein farnesylation would sensitize MCF-7 cells to the action of ROSC. MCF-7 cells were treated with 1-40 microM ROSC alone, or in combination with L-744,832, an inhibitor of farnesyl protein transferase (FTPase). To measure the impact on the proliferation of the cells, we used the CellTiterGlo viability assay and FACS analysis was employed to quantify the DNA-content of the single cells. The amount and phosphorylation status of relevant proteins after lysis of MCF-7 cells was assessed on Western blots using (phospho)-specific antibodies. The combined treatment with L-744,832 and ROSC for 24 h, markedly reduced the number of viable MCF-7 cells, primarily, by re-enforcing the cell cycle arrest. Interestingly, the potentiation of the ROSC-mediated inhibition of cell proliferation became evident during the 48 h post-incubation period in presence of the FPTase inhibitor. Inhibition of FPTase in ROSC-treated cells reduced the number of viable cells by approximately 30%. Evidently, the combined treatment sensitizes MCF-7 cells to the action of ROSC, thereby allowing to reduce the dose of the drug and to minimize side effects.

The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1.

FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.

Interplay between the p53 tumor suppressor protein family and Cdk5: novel therapeutic approaches for the treatment of neurodegenerative diseases using selective Cdk inhibitors.

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as tau protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.

Prevention of farnesylation of c-Ha-Ras protein enhances synergistically the cytotoxic action of doxorubicin in cycling but not in quiescent cells.

Ras, the product of a proto-oncogene, is a GTP-hydrolyzing enzyme found mutated in approximately 50% of human cancers. "Gain of function" mutations of Ras lead to an escape of transformed cells from cell-cycle control, rendering them independent to stimulation by growth factors, giving them almost unlimited proliferation capacity. The cytosolic precursor isoform of Ras is biologically inactive. After several post-translational modifications, Ras is anchored to the plasma membrane and, thereby, the protein becomes activated. The finding that lipid modifications of Ras protein, particularly farnesylation, are essential for its signal transduction activity, gave rise to the concept that blocking farnesyl protein transferase (FPTase), the enzyme catalyzing the first step in the Ras modification cascade, would prevent proper membrane anchoring and provide an improved approach in the cure of tumors harboring Ras mutations. In the present study we used transformed rat cells overexpressing a temperature-sensitive p53 protein, adopting wt conformation at 32 degrees C and mutant conformation at 37 degrees C. We treated the cells growing at 32 or 37 degrees C with doxorubicin alone, or in combination with inhibitors of FPTase. Combined treatment was more efficient and the same inhibition of cell proliferation was reached at lower DOX concentrations. The treatment strongly affected the growth rate of tumor cells but only negligibly of normal cells. However, the inhibitors of FPTase prevented the membrane anchoring in both situations. These results show two striking advantages of the combined treatment: the desired cytostatic effect on tumor cells at lower drug concentrations and clearly reduced adverse effects on quiescent cells.

Dual action of the inhibitors of cyclin-dependent kinases: targeting of the cell-cycle progression and activation of wild-type p53 protein.

The inhibition of cyclin-dependent kinases (CDKs) represents a novel approach to the therapy of human malignancies. Already in clinical trials, recently developed CDK inhibitors very efficiently target the rapidly proliferating cancer cells and inhibit their cell-cycle progression. Interestingly, some CDK inhibitors additionally affect the stability and activity of the tumour-suppressor protein p53, thereby enhancing their antiproliferative action towards cancer cells. Considering the fact that the p53 protein is mutated or inactivated in approximately 50% of all human cancers, the efficacy of CDK inhibitor therapy could differ between cancer cells depending on their p53 status. Moreover, recent reports demonstrating that some cancer cells can proliferate despite CDK2 inhibition questioned the central role of CDK2 in the cell-cycle control and suitability of CDK2 as a therapeutic target; however, the p53 activation that is mediated by CDK inhibitors could be essential for the efficacy of CDK inhibitors in therapy of CDK2-independent cancers. Furthermore, there is also reason to believe that CDK2 inhibitors could be used for another purpose, to protect normal cells from the effects of chemotherapy.

Biochemical monitoring of mTOR inhibitor-based immunosuppression following kidney transplantation: a novel approach for tailored immunosuppressive therapy.

BACKGROUND: Immunosuppressive therapy with the mammalian target of rapamycin (mTOR) inhibitors requires a fine balance between allograft maintenance and drug-related side effects. METHODS: In this study we examined the feasibility of monitoring TOR inhibitor-based immunosuppression by assessment of the phosphorylation status at the Thr(389) site of the p70S6 kinase in peripheral blood mononuclear cells (PBMCs). At total of 36 patients with renal transplants and 8 healthy controls were enrolled. RESULTS: We found that sirolimus treatment was associated with a pronounced inhibition of p70S6 kinase phosphorylation, as compared to healthy donors or otherwise immunosuppressed patients. In sirolimus-treated patients, phosphorylation of the p70S6 kinase was significantly inhibited when sirolimus trough levels were > 6 ng/mL. In contrast, for trough levels <6 ng/mL, the degree of inhibition of p70S6 kinase phosphorylation showed a high degree of interindividual variability. We recorded a total of five clinical relevant rejection episodes in this patient category. Intriguingly, rejecters uniformly maintained a high degree of phosphorylation independent of the sirolimus trough level whereas non-rejecters showed a significant inhibition of phosphorylation. CONCLUSION: Therefore, the phosphorylation status of the p70S6 kinase appears to provide more relevant information on the desired effect of sirolimus in target cells as compared to trough level measurements. Moreover, this assay provides an opportunity to safely titer down sirolimus levels to avoid overimmunosuppression and, on the other hand, to identify patients with insufficient TOR inhibitor therapy that are at risk for rejection.

The subcellular distribution of the p53 tumour suppressor, and organismal ageing.

The p53 protein, the product of a tumour suppressor gene, is a key regulator of cell growth, differentiation and apoptosis. It is able to induce a transient cell cycle arrest and terminal senescence. Most of its functions are exerted by the transcriptional activation of genes involved in cell cycle control, DNA repair and apoptosis. The activation of p53 is primarily mediated by post-translational modifications that affect its conformation and capacity to bind to several proteins, resulting in its stabilization and enhanced DNA-binding potential. Another way to regulate the biological function of p53 involves changes in its intracellular distribution. This paper presents an overview of the role of p53 in cellular senescence and the regulation of p53 activity by its intracellular distribution.

Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1.

We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.

Rapamycin induces tumor-specific thrombosis via tissue factor in the presence of VEGF.

Therapeutic strategies that target and disrupt the already-formed vessel networks of growing tumors are actively pursued. The goal of these approaches is to induce a rapid shutdown of the vascular function of the tumor so that blood flow is arrested and tumor cell death occurs. Here we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin, when administered to tumor-bearing mice, selectively induced extensive local microthrombosis of the tumor microvasculature. Importantly, rapamycin administration had no detectable effect on the peritumoral or normal tissue. Intravital microscopy analysis of tumors implanted into skinfold chambers revealed that rapamycin led to a specific shutdown of initially patent tumor vessels. In human umbilical vein endothelial cells vascular endothelial growth factor (VEGF)-induced tissue factor expression was strongly enhanced by rapamycin. We further show by Western blot analysis that rapamycin interferes with a negative feedback mechanism controlling this pathologic VEGF-mediated tissue factor expression. This thrombogenic alteration of the endothelial cells was confirmed in a one-step coagulation assay. The circumstance that VEGF is up-regulated in most tumors may explain the remarkable selectivity of tumor vessel thrombosis under rapamycin therapy. Taken together, these data suggest that rapamycin, besides its known antiangiogenic properties, has a strong tumor-specific, antivascular effect in tumors.

Nbn heterozygosity renders mice susceptible to tumor formation and ionizing radiation-induced tumorigenesis.

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive disease characterized by microcephaly, growth retardation, immunodeficiency, chromosomal instability, and predisposition to cancer. Heterozygous NBS patients show increased chromosomal instability and are suspected to be at a high risk for cancer. To study the impact of NBS1 heterozygosity on malignancy susceptibility, we disrupted the murine homologue (Nbn) of NBS1 in mice using gene targeting techniques. While null mutation in the Nbn gene resulted in embryonic lethality at the blastocyst stage because of growth retardation and increased apoptosis, heterozygous knockout (Nbn(+/-)) mice developed a wide array of tumors affecting the liver, mammary gland, prostate, and lung, in addition to lymphomas. Moreover, gamma-irradiation enhanced tumor development in Nbn(+/-) mice, giving rise to a high frequency of epithelial tumors, mostly in the thyroid and lung, as well as lymphomas. These mice also developed numerous tumors in the ovary and testis. Southern and Western blot analyses showed a remaining wild-type allele and nibrin expression in Nbn(+/-) tumors. Sequencing analysis confirmed no mutation in the Nbn cDNA derived from these tumors. Cytogenetic analysis revealed that primary Nbn(+/-) embryonic fibroblasts and tumor cells exhibit increased chromosomal aberrations. These data suggest that haploinsufficiency, not loss of heterozygosity, of Nbn could be the mechanism underlying the tumor development. Taken together, our heterozygous Nbn-knockout mice represent a novel model to study the consequences of NBS1 heterozygosity on tumor development.