Anthony P. Albino

Neutral endopeptidase 24.11 loss in metastatic human prostate cancer contributes to androgen-independent progression

Neutral endopeptidase 24.11 (NEP) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). We report that NEP expression and catalytic activity are lost in vitro in androgen-independent but not androgen-dependent PC cell lines. In vivo, NEP protein expression is commonly decreased in cancer cells of metastatic PC specimens from patients with androgen-independent but not androgen-dependent PC. Overexpression of NEP in androgen-independent PC cells or incubation with recombinant NEP inhibits PC cell growth. Furthermore, in androgen-dependent PC cells, expression of NEP is transcriptionally regulated by androgen and decreases with androgen withdrawal. These data suggest that decreased NEP expression, common in androgen-independent PCs, is facilitated by the elimination of androgens, and that NEP loss plays an important role in the development of androgen-independent PC by allowing PC cells to use mitogenic neuropeptides as an alternate source to androgen in order to stimulate cell proliferation.

Cytometry of ATM Activation and Histone H2AX Phosphorylation to Estimate Extent of DNA Damage Induced by Exogenous Agents

This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double‐strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis‐a‐vis cell cycle phase position and induction of apoptosis that employ flow‐ and laser scanning‐cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide‐releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow‐ and image‐analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis.

Mutation and expression of the p53 gene in human malignant melanoma.

Derangement of the p53 tumor suppressor gene has been implicated in the aetiology of a wide range of human neoplasias. We have previously determined that overexpression and mutation of the p53 gene in cultured metastatic melanomas is low (11%). However, two recent immunohistochemical studies have reported that >85% of malignant melanoma specimens overexpress mutated p53 protein. In an effort to resolve this contradiction in the published literature, we have re-evaluated a range of cultured and non-cultured melanocytic lesions for the occurrence of point mutations in the p53 gene using DNA- and RNA-dependent single strand conformation polymorphism (RNA-SSCP) and direct DNA sequencing of polymerase chain reaction (PCR) amplified DNA, and overexpression of the p53 protein using immunohistochemistry. We found point mutations in 25% (9 of 36) of cultured melanomas and 0% in 34 fresh melanoma biopsies; however, increased p53 expression was found in 42% of paraffin-embedded melanoma specimens and 7% of benign lesions. The low frequency of p53 point mutations and high frequency of p53 expression suggests that derangement of the p53 gene by point mutations is not a common perturbation in the majority of melanoma cells, and that overexpression of p53 in this tumour type is due to a mechanism other than point mutation.

Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells

BackgroundAlthough lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimers and Parkinsons syndromes, and cancer.MethodsGene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry.ResultsWe show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2α) or phosphorylation (i.e., phospho-eIF2α) in a majority of human lung cancers.ConclusionThese data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2α and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue.

Cell‐surface peptidases are ectoenzymes which regulate the access of bioactive peptides to their receptors on cell membranes. Abnormalities in their expression and function result in altered peptide activity which contribute to neoplastic transformation and/or progression.

Antiproliferative and Antitumor Effects of α-Interferon in Renal Cell Carcinomas: Correlation with the Expression of a Kidney-associated Differentiation Glycoprotein

Human leukocyte alpha-interferon (IFN-alpha) has significant antitumor activity in advanced renal cell carcinoma (RC), with approximately 15% (range, 5 to 29%) of patients subjected to IFN-alpha therapy exhibiting a major objective response. We assayed 16 RC cell lines for intrinsic sensitivity to the growth-inhibitory effects of recombinant IFN-alpha. Similar to results observed in patients, cultured RCs could be divided into those that are inhibited by IFN-alpha and those that are not. In addition, the IFN-alpha-sensitive or -resistant phenotype of cultured RCs was correlated with surface expression of six unrelated kidney-associated differentiation antigens. The expression of one antigen, a Mr 160,000 glycoprotein (gp160), was found to correlate with resistance to IFN-alpha. Proliferation of seven RC cell lines expressing gp160 (gp160+) was not significantly inhibited by IFN-alpha at concentrations as high as 3000 units/ml. In contrast, proliferation of eight of nine RC cell lines lacking expression of gp160 (gp160-) was markedly inhibited by IFN-alpha. The effect of IFN-alpha on gp160+ and gp160- RC xenografts in nu/nu mice was examined. In separate experiments, two gp160+ RC cell lines and five gp160- RC cell lines were injected s.c. into nu/nu mice; one half of the mice were subsequently treated with 10(6) units of IFN-alpha i.p. 3 times a wk, and one half received no IFN-alpha. Tumors appeared at the sites of inoculation in all mice given injections of gp160+ RC cell lines within 10 to 25 days regardless of INF-alpha therapy. Mice given injections of gp160- RC cell lines, but not receiving IFN-alpha, also formed tumors. In contrast, gp160- RC cell lines injected into mice that were treated with IFN-alpha exhibited a marked sensitivity, as demonstrated by either no tumor formation or delayed tumor formation. We conclude that the absence of gp160 expression by RCs may be predictive of sensitivity to the antitumor effects of IFN-alpha and, thus, provide a basis for identifying IFN-responsive patients.

Induction of H2AX Phosphorylation in Pulmonary Cells by Tobacco Smoke A New Assay for Carcinogens

DNA double strand breaks (DSBs) are potentially carcinogenic lesions. The induction of DSBs triggers phosphorylation of histone H2AX. Phosphorylated H2AX, denoted p-H2AX, may be detected immunocytochemically and the intensity of p-H2AX immunofluorescence (IF) reveals the frequency of DSBs. Using this assay we tested whether the exposure of A549 human pulmonary adenocarcinoma cells to tobacco smoke, and normal human bronchial epithelial cells (NHBE) to tobacco smoke condensate, induces DSBs. Cellular p-H2AX IF and DAPI fluorescence of individual cells were measured by laser scanning cytometry (LSC). Exposure of A549 cells to tobacco smoke and NHBE cells to smoke condensate led to H2AX phosphorylation in both a time and dose dependent manner. The maximal rate of H2AX phosphorylation was seen during the initial 4h of cell treatment. At high doses (50 _g/ml of smoke condensate), H2AX phosphorylation continued to increase for up to 24h. No differences in the level of H2AX phosphorylation were apparent between cells in G1 vs S vs G2/M phase of the cell cycle in response to treatment with smoke condensate. The data provide strong evidence that exposure of A549 cells to tobacco smoke or NHBE cells to smoke condensate rapidly induces DSBs in these cells. The present assay to detect and measure DSBs induced by tobacco products complements other mutagenicity assays and may be applied to test potential carcinogens in other products.

Infrequent ras Oncogene Point Mutations in Renal Cell Carcinoma

The role of ras oncogenes in the pathogenesis of renal cell carcinoma is unclear. We have previously shown that insertion of a mutated ras oncogene into cultured human proximal tubular cells, the normal counterpart of renal cell carcinomas, initiates a series of transformation events which results in cells possessing a renal cancer phenotype. These data suggested a role for mutated ras genes in the initiation and maintenance of this disease. Therefore, to assess the involvement of ras genes in renal carcinogenesis, 51 primary and metastatic renal carcinomas, including three oncocytomas, were analyzed for point mutations in codons 12, 13 and 61 of the Ha-ras, Ki-ras and N-ras proto-oncogenes using polymerase-catalyzed chain reaction methodology. A mutated Ha-ras gene was found in one renal cancer metastatic to lung for an overall incidence of 2%. These data indicate that ras oncogenes, activated by point mutations, do not play a major role in the initiation, maintenance or metastases of renal carcinomas.

Serological response of melanoma patients to vaccines prepared from VSV lysates of autologous and allogeneic cultured melanoma cells

Melanoma patients were vaccinated with cell‐free lysates prepared from vesicular stomatitis virus (VSV)‐infected cultured autologous and allogeneic melanoma cells. Eleven patients received vaccines produced from the melanoma cell line SK‐MEL‐13. This cell line, derived from the melanoma of Patient AH, expresses a differentiation antigen (initially defined by autologous antibody) that is restricted to melanomas and other cells of neural crest origin (an example of a Class 2 melanoma antigen). Thirteen patients received vaccines prepared from autologous melanoma cells, the only known source of autologous unique (Class 1) melanoma antigens. VSV lysates were used for vaccination because VSV infection of tumor cells has been shown to augment the immunogenicity of tumor antigens. All patients but one vaccinated with VSV lysates of autologous melanoma cells developed antibodies against VSV, and all patients vaccinated with VSV lysates of SK‐MEL‐13 developed antibodies against HLA‐related antigens. Antibodies against a Class 1 (unique) melanoma antigen were detected in only one case, and antibodies against Class 2 (shared) melanoma antigens were not found in any of the patients. The authors conclude that VSV lysates of melanoma cells are not effective in increasing the serologic response of melanoma patients to Class 1 or 2 melanoma antigens.

Expression of the mast cell growth factor interleukin-3 in melanocytic lesions correlates with an increased number of mast cells in the perilesional stroma: implications for melanoma progression

The molecular events responsible for tumor progression in human cutaneous malignant melanoma remain unclear; however, critical to the process is the dysregulated proliferation of tumor cells and the development of new vascular channels which allow further growth and dissemination. Connective tissue mast cells (MC) have been implicated in tumor progression because they concentrate around tumors (including melanomas) prior to the formation of new blood vessels, and because they contain many chemical mediators, including basic fibroblast growth factor (bFGF), known to have mitogenic and angiogenic effects. Several MC chemotactic and mitogenic factors have been described including interleukin‐3 (IL‐3). In order to determine whether there is a differential expression of this MC chemotactic/mitogenic factor with tumor progression in vivo, we evaluated by immunohistochemistry 85 melanocytic lesions including primary invasive malignant melanoma (PIMM), melanoma in situ (MMIS), and ordinary intradermal benign melanocytic nevi (BMN) for expression of IL‐3. Nucleic acid in situ hybridization also was used to evaluate the melanocytic lesions for IL‐3‐specific mRNA transcripts. Intracellular IL‐3 protein was detected in 29/33 (88%) PIMM and 15/25 (60%) MMIS, but was not detected in any (0/27; 0%) BMN (p<0.0001). IL‐3‐specific mRNA transcripts were present in 3/4 PIMM and 2/10 MMIS in which IL‐3 protein was not identified, but were not detected in any BMN. IL‐3 mRNA or protein was not detected in normal melanocytes present in the perilesional epidermis of any of the specimens studied. Immunohistochemistry also was used to confirm the presence of IL‐3α‐specific receptors on human cutaneous MC. As demonstrated by others, a significantly increased number of MC was present in the perilesional stroma of PIMM and MMIS vis à vis BMN (p<0.0001). The results suggest that melanoma cells may attract MC in vivo by producing MC chemotactic/mitogenic factors such as IL‐3. The recruitment of MC and the subsequent release of their potent mitogenic and angiogenic factors such as bFGF may thus represent a tumor–host interaction which favors tumor progression.