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 microg/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 G(1) vs S vs G(2)/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.

Loss of expression of protein kinase C beta is a common phenomenon in human malignant melanoma: a result of transformation or differentiation?

As with most cancers, the aetiology of human cutaneous melanoma is likely to be multifactorial and to include the accumulation of irreversible alterations in an unknown number of genes. Elucidating this molecular progression necessitates both the identification of genetic perturbations at each clinically relevant stage, and the assessment of their impact on the normal melanocyte. The observation that the epidermal melanocyte, in contrast to metastatic melanoma cells, requires activation of the protein kinase C (PKC) pathway to facilitate growth in vitro indicates that one or more isoforms (or substrates) of this large and complex family of proteins are among those that undergo alteration during the development of malignant melanoma. Consequently, a number of studies have investigated the expression of various PKC family members in both melanocyte and melanoma cell lines, without a consensus of opinion as to which isoforms are of biological significance in melanoma development and progression. The present study involved a comprehensive evaluation of the PKC profile in normal melanocytes and in 16 metastatic melanoma cell lines. The results show that the major difference in isoform expression between epidermal melanocytes and melanoma cells is the loss of PKCbeta protein expression in 90% of melanoma cell lines. Examination of PKCbeta in benign and malignant melanocytic lesions revealed that this protein is either downregulated or absent in both naevi and metastatic melanomas. We conjecture that, although the loss of PKCbeta expression is a common phenomenon in malignant melanocytes, it may be related more to a normal process of melanocytic differentiation than to malignant transformation.

Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation.

The incidence of cutaneous malignant melanoma is undergoing a dramatic increase in persons with light-color skin in all parts of the world. The prognosis for individuals with advanced disease is dismal due to the lack of effective treatment options. Thus, there is a need for new approaches to control tumor progression. Epidemiological, experimental, and mechanistic data implicate omega-6 polyunsaturated fatty acids (PUFAs) as stimulators and long-chain omega-3 PUFAs as inhibitors of development and progression of a range of human cancers, including melanoma. The aim of this study was to assess the mechanisms by which docosahexaenoic acid (DHA), an omega-3 PUFA, affects human melanoma cells. Exponentially growing melanoma cell lines were exposed in vitro to DHA and then assessed for (a) inhibition of cell growth; (b) expression of cyclins and cyclin-dependent kinase inhibitors in individual cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to cyclin D1, cyclin E, p21WAF1/CIP1, or p27(KIP1); and (c) expression of total pRb(T) independent of phosphorylation state and hypophosphorylated pRb(P-) in fixed cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to pRb(T) or pRb(P-), respectively. After treatment with increasing concentrations of DHA, cell growth in a majority of melanoma cell lines (7 of 12) was inhibited, whereas in 5 of 12 cell lines, cell growth was minimally affected. Two melanoma cell lines were examined in detail, one resistant (SK-Mel-29) and one sensitive (SK-Mel-110) to the inhibitory activity of DHA. SK-Mel-29 cells were unaffected by treatment with up to 2 microg/ml DHA whether grown in the absence or presence of 1% fetal bovine serum (FBS). No appreciable change was observed in cell growth, cell cycle distribution, the status of pRb phosphorylation, cyclin D1 expression, or the levels of the cyclin-dependent kinase inhibitors p21 and p27. In contrast, SK-Mel-110 cell growth was inhibited by DHA with the cells accumulating either in G1 or S phase: 0% in SK-Mel-29 versus 13.3 or 41.2% in SK-Mel-110 in the absence or presence of FBS, respectively. In the absence of serum, considerable death occurred by apoptosis. In addition, DHA treatment resulted in increasing numbers of SK-Mel-110 cells (from 12 to >40%) expressing hypophosphorylated pRb, whereas the levels of cyclin D1 and p21 changed little. Expression of p27 in these cells increased >2.5 times when grown in the absence of FBS but not in the presence of 1% FBS. Thus, we show for the first time that DHA inhibits the growth of cultured metastatic melanoma cells. Furthermore, growth inhibition correlates with a quantitative increase in hypophosphorylated pRb in the representative sensitive melanoma cell line SK-Mel-110. Although multiple factors influence pRb phosphorylation, it appears that both cyclin D1 and p21 expression do not change in the presence of DHA, although p27 was strikingly increased in SK-Mel-110 cells in the absence of FBS. The fact that pRb became hypophosphorylated after exposure to DHA suggests a cross-talk mechanism between fatty acid metabolism and the pRb pathway. Determining the mechanism by which PUFAs can inhibit melanoma growth will be an important first step in the rational use of PUFAs as antitumor agents.

Update of diagnostic and prognostic markers in cutaneous malignant melanoma.

The biomolecules described in this article generally have been studied as possible diagnostic or clinically prognostic markers in the context of melanoma disease progression as measured by the gold standards of tumor thickness and development of metastasis. Most of the markers showed variations in expression phenotype only during the deeply invasive or metastatic stage of tumor progression and were thus predictive of clinical outcome only for these subgroups of patients. Some of the markers may have utility in identifying patients with deeply invasive primary tumors who are likely to develop metastasis and thus should receive earlier, more aggressive treatments. In addition, some of the markers may identify patients likely to respond better to a new type of therapy (e.g., anti-angiogenic therapy in a patient whose tumor is overexpressing VEGF or immunotherapy for a patient whose tumor is expressing high levels of MART-1). In the future, it will probably be possible to employ new techniques, such as laser-guided microdissection of tissues, to isolate individual melanocytes in order to identify the earliest stage-specific defects that contribute to an aggressive biological behavior. Identifying the subset of patients with superficially invasive melanomas who will develop metastatic disease will continue to provide a challenge.

Divergent cellular differentiation pathways during the invasive stage of cutaneous malignant melanoma progression.

Melanocytic nevus cells in the dermis adopt many morphological features of Schwann cells. These differentiation-related changes typically are not observed in melanomas. However, nevus cells do not fully recapitulate a Schwann cell phenotype, because they lack expression of mature myelin-associated proteins. In this study, melanocytic nevi and malignant melanomas were examined by immunohistochemistry for expression of low-affinity nerve growth factor receptor (p75NGFR), neural cell adhesion molecule (CD56/N-CAM), and growth-associated phosphoprotein-43 (GAP-43). These three proteins define the earliest stages of Schwann cell development but are not expressed in myelinated Schwann cells or normal melanocytes. p75NGFR was expressed in 25 of 25 (100%) and CD56/N-CAM and GAP-43 in 23 of 25 (92%) nevi, predominantly in type C nevus cells and nevic corpuscles. Most (84%) of the nevi expressed all three proteins. In primary invasive and metastatic melanoma, expression of each of the three proteins was limited to

A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells.

Dipeptidyl peptidase IV (DPPIV) is a cell surface peptidase expressed by normal melanocytes, epithelial cells, and other cells. Malignant cells, including melanomas and carcinomas, frequently lose or alter DPPIV cell surface expression. Loss of DPPIV expression occurs during melanoma progression at a stage where transformed melanocytes become independent of exogenous growth factors for survival. Tetracycline-inducible expression vectors were constructed to express DPPIV in human melanoma cells. Reexpressing DPPIV in melanoma cells at or below levels expressed by normal melanocytes induced a profound change in phenotype that was characteristic of normal melanocytes. DPPIV expression led to a loss of tumorigenicity, anchorage-independent growth, a reversal in a block in differentiation, and an acquired dependence on exogenous growth factors for cell survival. Suppression of tumorigenicity and reversal of a block in differentiation were dependent on serine protease activity, assessed using mutant DPPIV molecules containing serine-->alanine substitutions. Surprisingly, dependence on exogenous growth factors was not dependent on serine protease activity. Reexpression of either wild-type or mutant DPPIV rescued expression of a second putative cell surface serine peptidase, fibroblast activation protein alpha, which can form a heterodimer with DPPIV. This observation suggests that rescue of fibroblast activation protein alpha may play a role in regulating growth of melanocytic cells. These results support the view that downregulation of DPPIV is an important early event in the pathogenesis of melanoma.

Update of diagnostic and prognostic markers in cutaneous malignant melanoma.

The biomolecules described in this article generally have been studied as possible diagnostic or clinically prognostic markers in the context of melanoma disease progression as measured by the gold standards of tumor thickness and development of metastasis. Most of the markers showed variations in expression phenotype only during the deeply invasive or metastatic stage of tumor progression and were thus predictive of clinical outcome only for these subgroups of patients. Some of the markers may have utility in identifying patients with deeply invasive primary tumors who are likely to develop metastasis and thus should receive earlier, more aggressive treatments. In addition, some of the markers may identify patients likely to respond better to a new type of therapy (e.g., anti-angiogenic therapy in a patient whose tumor is overexpressing VEGF or immunotherapy for a patient whose tumor is expressing high levels of MART-1). In the future, it will probably be possible to employ new techniques, such as laser-guided microdissection of tissues, to isolate individual melanocytes in order to identify the earliest stage-specific defects that contribute to an aggressive biological behavior. Identifying the subset of patients with superficially invasive melanomas who will develop metastatic disease will continue to provide a challenge.

Correlation of overexpression of the low-affinity p75 neurotrophin receptor with augmented invasion and heparanase production in human malignant melanoma cells.

The role of growth factor receptors in regulating the progression of human melanocytes toward tumorigenicity and ultimately a malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix (ECM)-degradative enzymes in melanoma cells remain undefined at the molecular level. The low-affinity p75 neurotrophin receptor (p75NTR), a cysteine-rich transmembrane glycoprotein, is frequently expressed in advanced stages of human melanoma, but the biological consequences of this expression are unknown. p75NTR can enhance the invasive potential of brain-metastatic melanoma cells in vitro. We have extended here these results and related the level of p75NTR in human metastatic melanoma cells to their invasive potential to target organs other than brain. Fluorescence activated cell sorting (FACS) analysis showed that 3 melanoma cell lines (SK-MEL-146, SK-MEL-119, 70W) had differential p75NTR contents, whereas SK-MEL-147 cells had elevated amounts of p75NTR. Two other melanoma cell lines (SK-MEL-94, SK-MEL-110) with point mutations in the p75NTR transmembrane domain had reduced (SK-MEL-94) or absent (SK-MEL-110) p75NTR. We also examined these cell lines for presence of TrkA receptor, the high-affinity receptor for nerve growth factor (NGF), the prototypic neurotrophin. No TrkA receptor expression was detected in any of the cell lines. The extent of p75NTR expression correlated with the capability of NGF to promote cellular invasion and with production of heparanase, an important ECM-degradative enzyme. Melanoma cells sorted for high p75NTR expression (p75NTR-H cells) had markedly greater (9- to 13-fold increase) invasive capabilities in response to NGF exposure than those sorted for low p75NTR expression (p75NTR-L cells). Additionally, NGF induced a 8- to 10-fold increase of heparanase activity in p75NTR-H cells. Thus, we propose that p75NTR-mediated trophic support profoundly affects melanoma cell invasion to neurotrophin-rich organs.

Chemotherapy and P-glycoprotein expression in chondrosarcoma.

Chondrosarcomas are alleged to be resistant to chemotherapy. A retrospective review of our experience primarily with dedifferentiated chondrosarcomas treated with chemotherapy was performed to reevaluate the efficacy of chemotherapy for this tumor. There were 18 patients: 14 stage IIB and four stage III. Seventeen patients had dedifferentiated chondrosarcoma. The median age at diagnosis was 57 years. Fourteen of the patients underwent wide excision of the tumor, two underwent amputation, and two had no surgery. The femur and the pelvis were the most common locations of the primary tumor. Chemotherapy for 11 of the patients consisted of cisplatin and doxorubicin. Survival was analyzed with the Kaplan-Meier method; the median survival was 12 months. The hypothesis that chondrosarcomas express P-glycoprotein was tested. Expression of P-glycoprotein was evaluated by immunostaining with use of the C494 and C219 antibodies on 41 benign and malignant cartilage tumors, six of which were from the patients in the chemotherapy group. Immunostaining revealed that 37 of 41 cartilage tumors expressed P-glycoprotein. The rate of survival of patients with high-grade chondrosarcoma treated with chemotherapy is poor. P-glycoprotein expression is common in benign and malignant cartilage lesions. The lack of response to chemotherapy may be related to the expression of P-glycoprotein.

p53 mutations in chondrosarcoma.

Chondrosarcoma is a primary bone tumor that has several different grades and variants. We evaluated 48 chondrosarcomas for p53 overexpression and p53 mutations. p53 expression was evaluated with immunohistochemistry using monoclonal antibodies PAb421, PAb1801, and PAb240. p53 mutations were identified with single-strand conformational polymorphism (SSCP) and DNA sequencing in selected cases. Immunohistochemistry revealed nuclear staining with PAb421 and PAb1801 in the spindle cell portion of one dedifferentiated chondrosarcoma. SSCP analysis was abnormal only in the case with positive immunostaining and localized the mutation to exons 7 and 8. DNA sequence analysis identified a point mutation of G to C in codon 276, resulting in an amino acid substitution of proline for alanine. This point mutation has been reported previously in other tumors but not in chondrosarcoma. Assimilation of our results with previous studies suggests that p53 mutations are present in a minority of chondrosarcomas but when present, are in higher grade chondrosarcomas and their variants.

Aminopeptidase A expression and enzymatic activity in primary human renal cancers.

The gp160 human kidney differentiation antigen is identical to human aminopeptidase A (APA), a zinc-dependent cell-surface metallopeptidase which hydrolyzes peptides with N-terminal acidic residues. GP160/APA is constitutively expressed by proximal tubule cells, the normal cellular counterpart of most renal cancers (RCs). Immunohistochemical analysis of gp160/APA protein expression in 62 primary renal tumor specimens using monoclonal antibody S4 revealed heterogeneous or homogeneous expression of gp160/APA in 46/51 (90%) of clear cell carcinomas in contrast with 1/8 (13%) papillary renal tumors and 0/3 oncocytomas (p<0.001). Analysis of five primary clear cell carcinomas for gp160 protein expression immunohistochemically and associated APA catalytic activity revealed one tumor which expressed gp160/APA protein which was enzymatically inactive. Direct sequence analysis of DNA derived from this specimen could not detect mutations within the zinc-binding domain which would eliminate gp160/APA catalytic activity. These data indicate that the gp160/APA protein is expressed by primary clear cell but not papillary RCs or oncocytomas, and that alterations in gp160/APA protein including loss of protein expression or enzymatic activity occur in 20% of primary clear cell RCs.

Increased numbers of mast cells in human middle ear cholesteatomas: implications for treatment.

HYPOTHESIS: Because many of the biologic phenomena in which mast cells are involved also are observed in human cholesteatoma pathology, the authors hypothesized that mast cells may play a role in this disease. The first test of this hypothesis is to determine whether there are an increased number of mast cells associated with cholesteatomas. BACKGROUND: The molecular and cellular defects that result in the pathologic features observed in acquired and congenital cholesteatomas are unknown. One common feature of cholesteatoma pathogenesis is the presence of bacteria and a numerous inflammatory cytokines expressed by host inflammatory cells. The interactions between inflammatory cells and cholesteatoma epithelium could result in the induction of other aberrant biologic features of cholesteatomas. Thus, it is critical to the understanding of the pathogenesis of cholesteatomas to define the specific role of each cell type involved in this disease. Connective tissue mast cells have a complex retinue of functions mediated via the secretion of a variety of cytokines and proteinases, and many of the biologic phenomena in which mast cells are involved also are observed in cholesteatoma pathology. METHODS: The authors evaluated by immunohistochemistry 36 cholesteatomas of all types (e.g., primary and secondary acquired, recurrent, and congenital) and 23 specimens of normal tissues (e.g., tympanic membrane, canal wall skin, and postauricular skin) for the expression of tryptase, a mast cell-specific protease. RESULTS: Cholesteatomas showed approximately threefold to sevenfold increase in the concentration of mast cells when compared with that of normal tissues. In addition, 19-34% of the mast cells were found within the suprabasal layers of the squamous epithelium of cholesteatoma subgroups, a phenomenon observed only in grossly inflamed tympanic membrane specimens, but not in other control tissues including minimally inflamed tympanic membranes. CONCLUSIONS: The authors conclude from these data that mast cells may represent a previously unrecognized host inflammatory cell, which plays an important role in the development of one or more traits of cholesteatoma pathology.

Expression of the kidney-associated differentiation glycoprotein gp160 and resistance to the antitumor effects of interferon alpha in renal cell carcinomas.

BACKGROUND: Alpha interferon (IFN-alpha) is commonly used to treat patients with advanced renal cell carcinoma (RCC). We previously reported that resistance of RCCs to IFN-alpha in vitro correlated with the expression of a cell-surface glycoprotein of 160,00 kD molecular weight (gp160) which we subsequently identified as aminopeptidase A. MATERIALS AND METHODS: To directly test the role of gp160/APA in IFN-resistance, we stably introduced the gp160/APA cDNA into IFN-sensitive SK-RC-49 cells resulting in the expression of an enzymatically active gp160/APA protein. In addition, to determine if gp160/APA expression could function as a marker of IFN-resistance in vivo, we assessed gp160/APA protein levels in autologous normal kidney and primary renal cancer specimens from 29 patients half of which were randomized to receive adjuvant IFN-alpha therapy following nephrectomy. RESULTS: Four clones which possessed varying amounts of gp160/APA specific enzyme activity were assayed for sensitivity to the antiproliferative effects of IFN-alpha. All four clones exhibited sensitivity to IFN-alpha similar to that observed with parental SK-RC-49 cells. The analysis of tumor tissue detected no significant difference between the mean level of gp160/APA in tissue from control and IFN-alpha treated patients (1.33 A.U. versus 0.9981 A.U., p = 0.23); however, the mean gp160/APA level was significantly less in tumor tissue (mean = 1.15 A.U.) compared to normal tissue (mean = 2.15 A.U.; p < 0.00001). Within the IFN-alpha treated group, tumor gp160/APA levels did not correlate with the development of metastases or survival (p = 0.469). CONCLUSIONS: These data indicate that gp160/APA does not directly convey IFN-resistance to RCC cells and suggest that expression of gp160/APA in primary RCCs does not predict the benefit of IFN-alpha therapy.

The neuropeptide/mast cell secretagogue substance P is expressed in cutaneous melanocytic lesions.

Substance P (SP) is a neuropeptide found in both the central and peripheral nervous system. In the skin, SP-containing neurons stimulate the release of histamine from connective tissue mast cells (MC). SP also can potentiate neoangiogenesis and induce dermal fibrosis. MC-derived histamine has potent vasoactive effects, is angiogenic, and promotes tissue fibroplasia. In addition to histamine, MC contain many other angiogenic factors and a variety of cytokines, growth factors, and proteolytic enzymes implicated in tissue remodeling, and normal as well as tumor-associated neoangiogenesis. Many MC-derived factors, including histamine, can enhance melanoma cell growth directly. MC often concentrate around cutaneous melanomas which also frequently are associated with angiogenesis and peritumoral fibrosis. The precise mediators of these responses have not been well defined. We evaluated by immunohistochemistry cutaneous lesions representing stages of progression of malignant melanoma and its precursor lesions for the expression of SP. SP was expressed in 17/25 (68%) primary invasive malignant melanomas, 2/5 (40%) metastatic melanomas, 6/10 (60%) melanomas in situ, 7/12 (58%) atypical (dysplastic) nevi, and 4/10 (40%) spindle and epithelioid cell (Spitz) nevi, but was not detected in any (0/11, 0%) acquired benign melanocytic nevi (p<0.05). Invasive melanomas were immunolabeled in both the intraepidermal and the dermal components of the lesions. For those atypical and Spitz nevi which expressed SP, most of the immunoreactive melanocytes were located at the dermal-epidermal junction overlying areas of papillary dermal fibrosis. The results show differential expression of SP among cutaneous melanocytic lesions and suggest that the expression of this neuropeptide together with other factors may contribute to some of the host responses associated with these lesions.

Cholesteatoma: a molecular and cellular puzzle.

HYPOTHESIS: There are at least three possible molecular models of cholesteatoma pathogenesis. Cholesteatoma may arise as a result of 1) the induction of a preneoplastic or neoplastic transformation event; 2) a defective wound-healing process; and/or 3) a pathologic collision of the host inflammatory response, normal middle ear epithelium, and a bacterial infection. BACKGROUND: There have been a number of speculations concerning the factors that foster the development of cholesteatoma. Before resolving the molecular basis for the pathogenesis of cholesteatomas, it is important to present and test plausible models that could explain how a cholesteatoma becomes invasive, migratory, hyperproliferative, aggressive, and recidivistic. METHODS: The authors evaluated by various techniques (e.g., immunohistochemistry, flow cytometry, and image analysis) a large number of cholesteatomas of all types (e.g., primary and secondary acquired, recurrent, and congenital) and a range of normal tissues (tympanic membrane, canal wall skin, and postauricular skin) for the expression of various proteins (e.g., p53, ectopeptidases, tryptase) and for the presence of DNA aneuploidy. RESULTS AND CONCLUSIONS: The authors' published and unpublished studies to date support several suppositions concerning the pathology of cholesteatomas. First, cholesteatoma epithelium behaves more like a wound-healing process than a neoplasm. The available evidence to date does not indicate that cholesteatomas have inherent genetic instability, a critical feature of all malignant lesions. Second, the induction of hyperproliferative cells in all layers of the cholesteatoma epidermis implicates a potential idiopathic response to both internal events as well as external stimuli in the form of cytokines released by infiltrating inflammatory cells. Third, the presence of bacteria may provide a critical link between the cholesteatoma and the host, which prevents the cholesteatoma epithelium from terminating specific differentiation programs and returning to a quiescent state in which it becomes minimally proliferative, nonmigratory, and noninvasive. Fourth, none of our data suggest that there are any obvious molecular or cellular differences among the various types of cholesteatomas (e.g., primary and secondary acquired, recidivistic, and congenital). Continued research should delineate the precise molecular and cellular dysfunction involved in the pathogenesis of cholesteatomas and how this knowledge can be useful in the clinical management of cholesteatomas.

Expression of p53 protein in human middle ear cholesteatomas: pathogenetic implications.

BACKGROUND: Cholesteatoma is a destructive lesion of the middle ear or mastoid process or both. The molecular and cellular defects that result in the clinical hallmarks of acquired and congenital cholesteatomas, namely invasion, migration, uncoordinated proliferation, altered differentiation, aggressiveness, and recidivism, are unknown. Determining the existence of defects in the normal biology, biochemistry, and genetic complement of the major cellular constituents comprising a cholesteatoma (i.e., fibroblasts and keratinocytes) is critical to the understanding of the pathogenesis of cholesteatomas. It has been speculated that the development of human cholesteatomas is due, in part, to the altered control of cellular proliferation, which tilts the balance toward the aggressive, invasive growth of squamous epithelium within the middle ear. However, whether this altered control is due to defects in the mechanisms and underlying genes that control proliferation, or to cytokines released from infiltrating inflammatory cells, or to some other mechanism is unknown. The nuclear phosphoprotein p53 tumor suppressor gene plays a critical regulatory role in cell cycle control and apoptosis. In the current article, the authors have analyzed congenital, primary and secondary acquired, and recurrent cholesteatomas for the altered expression of p53 and Ki-67, a marker of active proliferation. METHODS: p53 and Ki-67 expression was determined by immunohistochemical assays using specific monoclonal antibodies. RESULTS: The authors' results indicate that p53 is elevated 9- to 20-fold in all cholesteatomas when compared to the expression of p53 in normal postauricular skin or tympanic membrane. However, there is no concomitant increase in Ki-67 expression in cholesteatomas. CONCLUSIONS: These data indicate a defect in cholesteatomas in the mechanisms that p53 engages (i.e., cell cycle control or apoptosis or both). In addition, these data further suggest that there is no intrinsic difference between any clinicopathologic group of cholesteatomas, at least with respect to p53-expression and, presumably, p53 function.

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.

Reversal of the low-affinity neurotrophin receptor stromal-epithelial expression pattern between benign and malignant human prostate.

Reduced expression of the low-affinity p75 neurotrophin receptor (p75(NTR)) occurs in prostate epithelial cells during malignant transformation. Recent studies indicating that the p75(NTR) can transduce signals that induce apoptosis suggest that diminished p75(NTR) in transformed prostate cells may contribute to immortalization. Mutations in the transmembrane domain of the p75(NTR) gene have been associated with decreased p75(NTR) protein expression and may block the ability of the p75(NTR) to induce apoptosis. Therefore, we used Western blot to analyze prostate cancer (PC) cell lines for p75(NTR) protein expression and gene single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing to analyze mutations in the transmembrane domain of the p75(NTR). p75(NTR) Protein was present in all cell lines, and mutations in the p75(NTR) gene were not detected in cDNA derived from any cell line. To define the expression pattern of p75(NTR) in PCs in vivo, we used immunohistochemical techniques to examine tissue specimens from 20 benign, 19 malignant primary, and 14 metastatic prostate specimens. In benign prostate tissues, expression of p75(NTR) was universally detected in basal cells but not in secretory epithelial or stromal cells. In both primary and metastatic PC tissues, p75(NTR) immunoreactivity could not be detected in malignant prostate epithelial cells. However, in contrast to the benign prostate, p75(NTR) protein was expressed in stromal cells surrounding malignant epithelial cells. Stromal p75(NTR) expression was present in 84% (16 of 19) primary and in 86% (12 of 14) metastatic specimens. These data show that in the benign prostate p75(NTR) protein is expressed by basal cells and not stromal cells whereas in malignant prostate p75(NTR) protein is expressed by stromal cells but not prostatic carcinoma cells. Reversal of the p75(NTR) stromal-epithelial pattern of expression between benign and malignant prostate suggests that p75(NTR) may contribute to the development and maintenance of prostate cancer.

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

BACKGROUND: 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. METHODS: Expression of aminopeptidase A (APA), aminopeptidase N (APN, CD13), and dipeptidyl peptidase IV (DPP IV, CD26) was immunohistochemically examined in 20 benign and 33 malignant prostate tissues (19 primaries and 14 metastases). RESULTS: Benign prostatic stroma exhibited no APA, APN, or DPP IV immunoreactivity. Stromal cells surrounding prostatic carcinoma cells demonstrated increased APA expression in 24/33 (73%) of tumors. Benign prostatic epithelial cells strongly expressed APN and DPP IV but not APA. In contrast, APN was expressed in > 80% of tumor cells in 5/33 (15%) of specimens, heterogeneously expressed (20-80% of cells positive) in 4/33 (12%) of specimens, and minimally expressed or absent in 24/33 (73%) of tumor specimens, with a similar pattern of expression in primary and metastatic tumors. DPP IV was expressed by > 80% of tumor cells in 18/19 (95%) of primary prostate cancer specimens, but in only 7/14 (50%) of metastases. CONCLUSIONS: These data show that cell-surface peptidases are differentially expressed by normal prostatic stromal and epithelial cells, with increased expression of APA in the stroma surrounding prostate cancer cells, absent APN expression in most tumor cells, and a decreased frequency of DPP IV expression in metastatic tumors. Further studies will elucidate the biological effects of the presence or loss of cell-surface peptidases in the benign and malignant prostate.

Loss of heterozygosity and homozygous deletion of the tpr locus in human gastric cancer.

BACKGROUND: Despite being one of the world's most common neoplasias, there is little information on the molecular events that lead to gastric cancer. Molecular studies have shown that inactivation of tumor suppressor genes by mutation and/or allelic loss is an important genetic alteration in the multistep process of tumorigenesis. METHODS: In an attempt to identify a putative tumor suppressor gene involved in the carcinogenesis of gastric cancer, we performed Southern blot analysis using the tpr probe for 44 patients with gastric cancer, using tumor tissue and normal tissue from the same specimen. RESULTS: Of the 44 samples, 7 (16%) were informative, heterozygous cases for the tpr probe. Three of the informative cases showed a loss of heterozygosity and 3 cases showed homozygous deletion for the tpr probe (6 of 7; 85%). CONCLUSIONS: These findings suggest that tpr gene plays a role in gastric tumorigenesis, and this may be due to a tumor suppressor effect for the tpr gene.