Protecting normal cells from the cytotoxicity of chemotherapy

Comment on: van Leeuwen IMM, et al. Cell Cycle 2012; 11:1851-6

Stochastic Competition between Mechanistically Independent Slippage and Death Pathways Determines Cell Fate during Mitotic Arrest

Variability in cell-to-cell behavior within clonal populations can be attributed to the inherent stochasticity of biochemical reactions. Most single-cell studies have examined variation in behavior due to randomness in gene transcription. Here we investigate the mechanism of cell fate choice and the origin of cell-to-cell variation during mitotic arrest, when transcription is silenced. Prolonged mitotic arrest is commonly observed in cells treated with anti-mitotic drugs. Cell fate during mitotic arrest is determined by two alternative pathways, one promoting cell death, the other promoting cyclin B1 degradation, which leads to mitotic slippage and survival. It has been unclear whether these pathways are mechanistically coupled or independent. In this study we experimentally uncoupled these two pathways using zVAD-fmk to block cell death or Cdc20 knockdown to block slippage. We then used time-lapse imaging to score the kinetics of single cells adopting the remaining fate. We also used kinetic simulation to test whether the behaviors of death versus slippage in cell populations where both pathways are active can be quantitatively recapitulated by a model that assumes stochastic competition between the pathways. Our data are well fit by a model where the two pathways are mechanistically independent, and cell fate is determined by a stochastic kinetic competition between them that results in cell-to-cell variation

Src tyrosine kinase augments taxotere-induced apoptosis through enhanced expression and phosphorylation of Bcl-2

Activation of Src, which has an intrinsic protein tyrosine kinase activity, has been demonstrated in many human tumours, such as colorectal and breast cancers, and is closely associated with the pathogenesis and metastatic potential of these cancers. In this study, we have examined the effect of activated Src on the sensitivity to taxotere, an anticancer drug targeting microtubules, using v-src-transfected HAG-1 human gall bladder epithelial cells. As compared with parental HAG-1 cell line, v-src-transfected HAG/src3-1 cells became 5.9 and 7.0-fold sensitive to taxotere for 2 and 24-h exposure, respectively. By contrast, HAG-1 cells transfected with activated Ras, which acts downstream of Src, acquired approximately 2.5∼4.8-fold taxotere resistance. The taxotere sensitivity in HAG/src3-1 cells was reversed, if not completely, by herbimycin A, a specific inhibitor of Src family protein tyrosine kinase, indicating that Src protein tyrosine kinase augments sensitivity to taxotere. Treatment of HAG/src3-1 cells with taxotere resulted in phosphorylation of Bcl-2 and subsequent induction of apoptotic cell death, whereas neither Bcl-2 phosphorylation nor apoptosis occurred in parental or c-H-ras-transfected HAG-1 cells. Interestingly, the Bcl-2 protein is overexpressed in v-src-transfected cell line, compared to those in parental or Ras-transfected cell line. Treatment of HAG/src3-1 cells with herbimycin A significantly reduced the expression and phosphorylation of Bcl-2, and abrogated taxotere-induced apoptosis, suggesting a potential role for Src protein tyrosine kinase in the taxotere-induced apoptotic events. H-7, a protein kinase C inhibitor and wortmannin, a phosphatidylinositol-3 kinase (PI-3 kinase) inhibitor, neither altered taxotere sensitivity nor inhibited taxotere-induced apoptosis in these cells. These data indicate that the ability of activated Src to increase taxotere sensitivity would be mediated by apoptotic events occurring through Src to downstream signal transduction pathways toward Bcl-2 phosphorylation, but not by activated Ras, PI-3 kinase or protein kinase C

Differential effect of vinorelbine versus paclitaxel on ERK2 kinase activity during apoptosis in MCF-7 cells

The effects of vinorelbine and paclitaxel on the activity of extracellular signal-regulated protein kinase2 (ERK2), a member of MAP kinase, and its role in the induction of bcl-2 phosphorylation and apoptosis were evaluated in MCF-7 cells. We demonstrated that ERK2 was activated rapidly by vinorelbine, and was inhibited by either paclitaxel or estramustine. A 3-fold increase of ERK2 kinase activity was observed within 30 min when MCF-7 cells were treated with 0.1 μM vinorelbine. In contrast, the same treatment with paclitaxel resulted in a significant decrease of ERK2 kinase activity. We also demonstrated that elevated bcl-2 phosphorylation induced by vinorelbine is paralleled by decrease of a complex formation between bcl-2 and bax, cleavage of poly (ADP) ribose polymerase (PARP) protein, activation of caspase-7, and apoptosis. The levels of bcl-2 phosphorylation, bax, and PARP were not significantly affected by 2′-amino-3′-methoxyflavone (PD 98059), an ERK kinase specific inhibitor. Thus, our data suggest that the apoptosis induced by vinorelbine in MCF-7 cells is mediated through the bcl-2 phosphorylation/bax/caspases pathways, and that activation of ERK2 by vinorelbine does not directly lead to the drug-mediated apoptosis. Since decrease of PARP occurred quickly following the treatment of MCF-7 cells with either 0.1 μM of vinorelbine or paclitaxel, this protein may serve as an early indicator of apoptosis induced not only by DNA damaging agents, but also by antimicrotubule drugs.   http://www.bjcancer.com © 2001 Cancer Research Campaig

Infiltrating ductal and lobular breast carcinomas are characterised by different interrelationships among markers related to angiogenesis and hormone dependence

To obtain a more integrated understanding of the different breast cancer phenotypes and to investigate whether bio-molecular profiles can distinguish between specific histotypes, we explored the interrelations among several biologic variables indicative of, or related to, hormone dependence, proliferation and apoptosis control, and angiogenesis in ductal and lobular carcinomas, the most common histotypes. Oestrogen and progesterone receptors, tumour proliferative activity, the expression of cyclin A, p16ink4A, p27kip1, p21waf1, p53, bcl-2, and levels of vascular endothelial growth factor and hypoxia-inducible factor-1α (HIF-1α) were evaluated in 190 in ductal and 67 lobular carcinomas. Our findings support the hypothesis that in ductal and lobular carcinomas are two distinct, partially phenotypically unrelated entities, the latter being characterised by the presence of features indicative of differentiation such as oestrogen receptors, low proliferation and lack of p53 expression and associated with low vascular endothelial growth factor content compared to angiogenesis in ductal carcinomas. Conversely, no significant difference was found between lobular carcinomas and in ductal carcinomas considering the frequency distribution of PgR-positive cases, cyclin-dependent kinase inhibitors acting at the G1/S boundary, bcl-2 and HIF-1α protein expression. Although both generally defined as hormone responsive, in ductal and lobular carcinomas are also characterised by biologic patterns in which proteins related to hormone responsiveness, cell-cycle control, apoptosis and angiogenesis were differently associated. This finding suggests the need to refine breast cancer characterisation in order to provide detailed information about individual tumours, or subsets of tumours, that will help in defining optimal treatment approaches

Constitutive cytoplasmic localization of p21Waf1/Cip1 affects the apoptotic process in monocytic leukaemia

In the present study, we analysed the expression and localization of p21Waf1/Cip1 in normal and malignant haematopoietic cells. We demonstrate that in normal monocytic cells, protein kinase C (PKC)-induced p21 gene activation, which is nuclear factor-κB (NF-κB) independent, results in predominantly cytoplasmic localized p21 protein. In acute monocytic leukaemia (M4, M5), monocytic blasts (N=12) show constitutive cytoplasmic p21 expression in 75% of the cases, while in myeloid leukaemic blasts (N=10), low nuclear and cytoplasmic localization of p21 could be detected, which is also PKC dependent. Constitutive p21 expression in monocytic leukaemia might have important antiapoptotic functions. This is supported by the finding that in U937 cells overexpressing p21, VP16-induced apoptosis is significantly reduced (20.0±0.9 vs 55.8±3.8%, P<0.01, N=5), reflected by a reduced phosphorylation of p38 and JNK. Similarly, AML blasts with high cytoplasmic p21 were less sensitive to VP16-induced apoptosis as compared to AML cases with low or undetectable p21 expression (42.25 vs 12.3%, P<0.01). Moreover, complex formation between p21 and ASK1 could be demonstrated in AML cells, by means of coimmunoprecipitation. In summary, these results indicate that p21 has an antiapoptotic role in monocytic leukaemia, and that p21 expression is regulated in a PKC-dependent and NF-κB independent manner.

Signs of positive selection of somatic mutations in human cancers detected by EST sequence analysis

BACKGROUND: Carcinogenesis typically involves multiple somatic mutations in caretaker (DNA repair) and gatekeeper (tumor suppressors and oncogenes) genes. Analysis of mutation spectra of the tumor suppressor that is most commonly mutated in human cancers, p53, unexpectedly suggested that somatic evolution of the p53 gene during tumorigenesis is dominated by positive selection for gain of function. This conclusion is supported by accumulating experimental evidence of evolution of new functions of p53 in tumors. These findings prompted a genome-wide analysis of possible positive selection during tumor evolution. METHODS: A comprehensive analysis of probable somatic mutations in the sequences of Expressed Sequence Tags (ESTs) from malignant tumors and normal tissues was performed in order to access the prevalence of positive selection in cancer evolution. For each EST, the numbers of synonymous and non-synonymous substitutions were calculated. In order to identify genes with a signature of positive selection in cancers, these numbers were compared to: i) expected numbers and ii) the numbers for the respective genes in the ESTs from normal tissues. RESULTS: We identified 112 genes with a signature of positive selection in cancers, i.e., a significantly elevated ratio of non-synonymous to synonymous substitutions, in tumors as compared to 37 such genes in an approximately equal-sized EST collection from normal tissues. A substantial fraction of the tumor-specific positive-selection candidates have experimentally demonstrated or strongly predicted links to cancer. CONCLUSION: The results of EST analysis should be interpreted with extreme caution given the noise introduced by sequencing errors and undetected polymorphisms. Furthermore, an inherent limitation of EST analysis is that multiple mutations amenable to statistical analysis can be detected only in relatively highly expressed genes. Nevertheless, the present results suggest that positive selection might affect a substantial number of genes during tumorigenic somatic evolution