Giuseppe Starace

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.

Tumor necrosis factor α (TNF-α) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-α by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-α activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (2). To determine which MAPK signaling pathway is required for TNF-α induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-α receptors and both human and mouse TNF-α, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-α up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-α and IFN-γ markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-α-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-α and IFN-γ on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.

AKT Is Activated in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner in Response to Temozolomide and Confers Protection against Drug-Induced Cell Growth Inhibition

The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)- and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinase-dead form of AKT1 into breast cancer cells abrogated TMZ-induced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor d-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O6-guanine-methylating agents.

Mutant p53-induced Up-regulation of Mitogen-activated Protein Kinase Kinase 3 Contributes to Gain of Function

Mitogen-activated protein kinase kinase 3 (MAP2K3) is a member of the dual specificity kinase group. Growing evidence links MAP2K3 to invasion and tumor progression. Here, we identify MAP2K3 as a transcriptional target of endogenous gain-of-function p53 mutants R273H, R175H, and R280K. We show that MAP2K3 modulation occurred at the mRNA and protein levels and that endogenous mutant p53 proteins are capable of binding to and activate the MAP2K3 promoter. In addition, we found that the studied p53 mutants regulate MAP2K3 gene expression through the involvement of the transcriptional cofactors NF-Y and NF-κB. Finally, functional studies showed that endogenous MAP2K3 knockdown inhibits proliferation and survival of human tumor cells, whereas the ectopic expression of MAP2K3 can rescue the proliferative defect induced by mutant p53 knockdown. Taken together, our findings define a novel player through which mutant p53 exerts its gain-of-function activity in cancer cells.

The relevance of tumor size and cell kinetics as predictors of radiation response in head and neck cancer. A randomized study on the effect of intraarterial chemotherapy followed by radiotherapy

Size and extent of a primary tumor strongly influences its response to irradiation. In order to investigate the relevance of this statement a randomized study was carried out in which irradiation of advanced carcinoma of head and neck was compared with a combined treatment where preirradiation intraarterial chemotherapy was administered in order to reduce the extent of the tumor. Intra‐arterial chemotherapy (MTX 3–5 mg/day for 25–35 days) was given to 72 cases with lesions in the oral cavity, the oropharynx and maxillary antrum. Depending on response the patients were restaged and submitted to radiotherapy. In 68 other cases randomly allocated, radiotherapy only was given with the same modality and technique. After chemotherapy 21 cases with intraoral and 14 cases with maxillary antrum and oropharynx cancers were classified Stage I and II (0 and 23, respectively, before treatment). Results after 4 years follow up showed both local control and survival statistically better in the combined treatment in intraoral cancer where irradiation was delivered by a combination of external and interstitial technique. In the other cases no statistically significant difference was found. Amelioration of the results in the oral cavity could be ascribed to the greater use of interstitial irradiation due to the tumor shrinkage obtained with preirradiation chemotherapy more than to some synergistic effect between drug and irradiation. In 8 cases tumor cell kinetics were performed and the nuclear DNA content measured, but none of the kinetic parameters could be correlated with the response to treatment, even when the cancers were grouped according to their clinical growth characteristics. Late results in the irradiation of head and neck cancer probably depends on the presence of some resistant component which cannot be predicted by the cell kinetic or nuclear DNA content. Surgical excision or a second course of chemotherapy must be considered as a part of the multimodality treatment.

Significance of growth rates, cell kinetics, and histology in the irradiation and chemotherapy of squamous cell carcinoma of the mouth

In eight cases of extended tumors of oral cavity, we have studied the growth rate, the cell kinetics parameters, and the histology before and during treatment. We have found that growth rate and cell kinetics are not useful for clinical purposes because the first is not related to the response to treatment, while the second is unable to give relevant parameters in solid human tumors. New information was obtained from examination of serial biopsies before and throughout the treatment. These data suggest that multiple subpopulation groups of cancer cells with different intrinsic sensitivity to chemotherapy and/or radiotherapy may exist in the same tumor. Resistance of the tumor is related to some groups of cells scarcely sensitive to treatment.

The cyclin-dependent kinase inhibitor PHA-848125 suppresses the in vitro growth of human melanomas sensitive or resistant to temozolomide, and shows synergistic effects in combination with this triazene compound

PHA-848125 is a novel cyclin-dependent kinase inhibitor under Phase I/II clinical investigation. In this study, we describe, for the first time, the effect of PHA-848125 on human melanoma cells in vitro. Seven melanoma cell lines with different sensitivity to temozolomide (TMZ) were exposed to PHA-848125 for 5 days and then assayed for cell growth. In all cases, including TMZ-resistant cells, PHA-848125 IC(50) values were significantly below the maximum plasma concentrations achievable in the clinic. In the most PHA-848125-sensitive cell line, the drug caused a concentration-dependent G(1) arrest. PHA-848125 also impaired phosphorylation of the retinoblastoma protein at CDK2 and CDK4 specific sites, decreased retinoblastoma protein and cyclin A levels, and increased p21(Cip1), p27(Kip1) and p53 expression. Combined treatment with fixed ratios of TMZ plus PHA-848125 was studied in three melanoma cell lines. PHA-848125 was added to the cells 48 h after TMZ and cell growth was evaluated after 3 additional days of culture. Parallel experiments were performed in the presence of O(6)-benzylguanine (BG), to prevent repair of methyl adducts at O(6)-guanine induced by TMZ. Drug combination of TMZ plus BG and PHA-848125 produced additive or synergistic effects on cell growth, depending on the cell line. In the absence of BG, the combination was still more active than the single agents in the cell line moderately sensitive to TMZ, but comparable to PHA-848125 alone in the two TMZ-resistant cell lines. When TMZ plus BG were used in combination with PHA-848125 against cultured normal melanocytes, neither synergistic nor additive antiproliferative effects were observed. Our results indicate that PHA-848125 can have a therapeutic potential in melanoma patients, alone or combined with TMZ. Moreover this agent appears to be particularly attractive on the bases of its effectiveness against TMZ-resistant melanoma cells.

Cytokinetic evaluation in human head and neck cancer by autoradiography and DNA cytofluorometry

As part of a comprehensive research program on cell proliferation characteristics of squamous cell carcinoma in humans and the relation of these characteristics to treatment, 4 untreated cases of head and neck cancer and 2 recurrences were studied. Previously, cell kinetics following the percent labelled mitoses method were done and the same carcinomas now studied by measuring microfluorometrically the time distribution of the nuclear DNA content. Two cases were characterized by a homogeneous cell population, whereas in 2 other cases, two cycling subpopulations distinguished by their DNA content were encountered. The clinical response was not related to the occurrence of single‐ or multiple‐cell populations. In all cases, a great variability in the mitotic index (MI) and in the proportion of cells in the different cycle phases was found. The heterogeneity in the proliferative structure of these tumors stresses the need for a greater accuracy in performing cell kinetics studies in vivo and could account for the difficulties in devising a therapeutic regimen based on cycle or phase‐specific agents.