Overexpression of p53 in prostate carcinoma is associated with improved overall survival but not predictive of response to radiotherapy.

p53 gene mutations are among the most common specific genetic alterations in human cancer. Inactivation of p53 and subsequent protein accumulation has been implicated in a variety of human malignancies and associated with prostate cancer progression. In this study, we assessed p53 protein overexpression and gene mutations in prostate carcinoma and investigated associations between p53 alterations and clinicopathological parameters, survival, and response to radiotherapy. We evaluated 58 archival formalin-fixed, paraffin-embedded prostate carcinomas to detect abnormal p53 nuclear protein accumulation using immunohistochemistry. p53 mutational status of tumor DNA was evaluated using polymerase chain reaction-single-strand conformation polymorphism analysis of exons 5-9 and confirmed by direct DNA sequencing. Univariate and multivariate statistical analysis was used to determine the association of p53 status with clinical characteristics and response to radiotherapy. Overexpression of p53 was detected in 42 (72%) of 58 primary prostate carcinomas, but was undetectable in 7 samples of benign prostatic hyperplasias or 5 samples of normal prostate tissue. p53 exon 5-9 mutations were detected in 8 (14%) of 58 patient specimens. p53 mutational status, but not overexpression, was associated with higher Gleason scores (p=0.0145). Neither p53 overexpression nor mutation was associated with clinical stage, biochemical disease-free probability, or predictive of response to radiotherapy. p53 protein accumulation was inversely associated with improved overall survival (p=0.0108). Our studies demonstrate that p53 protein accumulation is a frequent alteration in prostate cancer. The disparity between p53 protein overexpression and p53 exon 5-9 mutations suggests the possibility of mutations outside this region or stabilization of wild-type p53 by alternative mechanisms. In our patient population, p53 protein overexpression or mutational status was not predictive of outcome in patients treated with radiation therapy. Additional studies are needed to further evaluate the association between p53 protein overexpression and improved overall survival.

Flt3-ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer.

We assessed the in vivo efficacy of Flt3-ligand (Flt3-L) treatment in C57BL/6 mice bearing a well-established MHC class I-negative prostate carcinoma TRAMP-C1. Flt3-L immunotherapy was initiated approximately 30 days after tumor inoculation, a time when > or =80% of the mice had palpable TRAMP-C1 tumors. Treatment with Flt3-L at 10 microg/day for 21 consecutive days suppressed TRAMP-C1 tumor growth and induced tumor stabilization (P = 0.0337). Enhanced tumor regression was demonstrated at a higher dose of 30 microg/day (P < 0.0001). Tumors excised from mice treated with Flt3-L were smaller than carrier-treated controls and contained a more pronounced mixed inflammatory cell infiltrate primarily composed of mphi. In regressor nice, tumors reappeared at the site of injection when Flt3-L therapy was terminated. When the experiment was repeated with MHC class I-positive TRAMP-C1 cells, tumor stabilization and/or regression was again observed after treatment (P < 0.0001); however, once again, tumors reappeared after the termination of therapy despite an extended treatment schedule (35 days). MHC class I-negative variants were present in tumors isolated from carrier- and Flt3-L-treated mice, and this phenotype could be reversed by IFN-gamma treatment in vitro. Thus, Flt3-L treatment of mice with preexisting transplantable prostate tumors results in tumor regression that is dose-dependent and accompanied by a pronounced mixed-cell inflammatory tumor infiltrate. However, disease relapse was invariably observed after the termination of therapy, which suggests that Flt3-L treatment of advanced MHC- prostate cancers will require adjuvant modalities to achieve a durable response.

Involvement of Shc in the signaling response of human prostate tumor cell lines to epidermal growth factor.

Autocrine growth factors for the epidermal growth factor receptor (EGFR) have been identified in prostate tumors, implicating a role for EGFR in the progression of prostate cancer. To investigate early signaling mechanisms used by the EGFR in prostate tumor cells, we have characterized the involvement of the Shc (src homology 2/x-collagen related) adapter protein in EGFR signaling in several human prostate tumor cell lines. In androgen-responsive lymph node-prostate cancer (LNCaP) cells and androgen-insensitive PC3, DU145 and PPC-I cells, Shc was identified as one of the most prominent phosphotyrosine proteins to be elevated in response to EGF. Equivalent levels of the 46- and 52-kDa Shc isoforms were detected in all of the tumor cell lines tested. However, levels of the 66-kDa isoform were variable among the cell lines. In all of the tumor cell lines, EGF caused an association between Shc and Grb2, another adapter protein linked to cellular ras activation. Additionally, several phosphotyrosine proteins, including a 115-120-kDa protein in EGF-treated LNCaP cells, co-associated with Shc. The profile of these Shc-associating proteins, however, differed among the tumor cell lines. Our results indicate that Shc is a common downstream element of EGFR signaling in prostate tumor cells and suggest multiple functions for Shc in prostate tumorigenesis.

Overexpression of EMS1/cortactin in NIH3T3 fibroblasts causes increased cell motility and invasion in vitro.

Cortactin, a p80/85 protein first identified as a src kinase substrate, is thought to be involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization. The cortactin gene, EMS1, maps to chromosome 11q13, a region amplified in head and neck squamous cell carcinomas (HNSCC) and breast cancer, which display lymph node metastasis and an unfavorable clinical outcome. To further address the role of cortactin in the malignant phenotype of cells, we stably overexpressed cortactin in NIH3T3 fibroblasts and evaluated the effects of elevated cortactin on cellular proliferation, motility and invasiveness. Cortactin overexpressing cells did not display any striking morphological changes, nor any significant differences in cell proliferation or saturation density as compared to control NIH3T3 cells. Furthermore, the cortactin overexpressing cells were anchorage dependent for growth. Interestingly, cortactin overexpressing cells were more motile and invasive in modified Boyden chamber assays. These results suggest that overexpression of cortactin may play a role in tumor progression by influencing tumor cell migration and invasion.

Antiproliferative and antiinvasive effects of carboxyamido-triazole on breast cancer cell lines.

BACKGROUND: Basement membrane invasion is one of the critical components of the metastatic cascade. The antiproliferative and antiinvasive activity of carboxyamido-triazole (CAI), a calcium influx inhibitor, was studied in five human breast cancer cell lines (MCF-7, MCF-7/ADRR, MDA-231, MDA-231R44, and BT-474). METHODS: Sensitivity of the cell lines to CAI was measured with a microculture tetrazolium assay. The Boyden chamber Matrigel chemoinvasion assay was used to measure the antiinvasive activity of CAI. Matrix metalloproteinase activity was analyzed by gelatin zymography. RESULTS: The 50% inhibitory concentrations of CAI were cell line dependent and ranged from 7.49 +/- 4.05 mumol/L to 46.1 +/- 8.6 mumol/L. CAI at a low, minimally toxic concentration (5 mumol/L) inhibited invasion by greater than 75% in the four invasive cell lines (MCF-7/ADRR, MDA-231, MDA-231R44, and BT-474) regardless of estrogen receptor or p-glycoprotein status (p < 0.01). CAI treatment also reduced matrix metalloproteinase activity in conditioned media from three of the four invasive lines (p < 0.05). CONCLUSIONS: CAI at clinically achievable concentrations is an effective antiproliferative and antiinvasive agent against human breast cancer cell lines regardless of estrogen receptor or p-glycoprotein status. Reduction in matrix metalloproteinase activity may be partially responsible for CAI inhibition of invasion.

Calcium signaling in prostate cancer cells: evidence for multiple receptors and enhanced sensitivity to bombesin/GRP.

BACKGROUND: Cellular calcium is an important second messenger for growth regulation. We sought to identify potentially important receptors on prostate tumor cells by screening over 20 agonists for their ability to increase intracellular free calcium ([Ca2+]i) in several human prostate tumor cell lines. METHODS: Intracellular calcium mobilization was detected using fura-2. RESULTS: We found bombesin, GRP, ATP/UTP, lysophosphatidic acid, thrombin, endothelin, histamine, and bradykinin increased [Ca2+]i in the advanced tumor cell lines DU-145, PC3, and PPC-1. Bombesin failed to elevate [Ca2+]i in an immortalized human prostate cell line. Rank-order of potency studies suggested the presence of P2U nucleotide receptors for ATP/UTP on prostate epithelial cells. Potency studies also revealed GRP > > bombesin > > neuromedin B at elevating [Ca2+]i in responding tumor cells. CONCLUSIONS: These findings indicate that androgen independent prostate tumor cell lines express multiple receptors capable of elevating intracellular calcium, and suggest that GRP receptors may be selectively expressed and/or coupled to calcium signaling during prostate tumor progression. Calcium sensitive cellular events may therefore contribute to the progression of prostate cancer.

Proposal: trauma as the cause of the Peyronie's lesion.

PURPOSE: We define the cause of the occurrence of Peyronie's disease. MATERIALS AND METHODS: Clinical evaluation of a large number of patients with Peyronie's disease, while taking into account the pathological and biochemical findings of the penis in patients who have been treated by surgery, has led to an understanding of the relationship of the anatomical structure of the penis to its rigidity during erection, and how the effect of the stress imposed upon those structures during intercourse is modified by the loss of compliance resulting from aging of the collagen composing those structures. Peyronie's disease occurs most frequently in middle-aged men, less frequently in older men and infrequently in younger men who have more elastic tissues. During erection, when full tumescence has occurred and the elastic tissues of the penis have reached the limit of their compliance, the strands of the septum give vertical rigidity to the penis. Bending the erect penis out of column stresses the attachment of the septal strands to the tunica albuginea. RESULTS: Plaques of Peyronie's disease are found where the strands of the septum are attached in the dorsal or ventral aspect of the penis. The pathological scar in the tunica albuginea of the corpora cavernosa in Peyronie's disease is characterized by excessive collagen accumulation, fibrin deposition and disordered elastic fibers in the plaque. CONCLUSIONS: We suggest that Peyronie's disease results from repetitive microvascular injury, with fibrin deposition and trapping in the tissue space that is not adequately cleared during the normal remodeling and repair of the tear in the tunica. Fibroblast activation and proliferation, enhanced vessel permeability and generation of chemotactic factors for leukocytes are stimulated by fibrin deposited in the normal process of wound healing. However, in Peyronie's disease the lesion fails to resolve either due to an inability to clear the original stimulus or due to further deposition of fibrin subsequent to repeated trauma. Collagen is also trapped and pathological fibrosis ensues.

Fibrin deposition in Peyronie's disease plaque.

PURPOSE: Peyronie's disease is a pathological fibrosis characterized by excessive deposition of collagen in the plaque. Although the etiology of Peyronie's disease is unknown, trauma has been hypothesized as the inciting event. In an effort to obtain more insight into the pathogenesis of Peyronie's disease plaque tissue was examined for collagen, elastic fiber, and fibrin content and distribution. MATERIALS AND METHODS: Plaque tissue specimens from 33 patients with Peyronie's disease, control penile tissue and nodular tissue from 8 patients with Dupuytren's contracture were analyzed histochemically for collagen staining and elastic fiber structure and distribution. Plaque tissue from 19 Peyronie's disease patients, control tissue and nodular tissue from Dupuytren's disease were also analyzed for the presence of fibrin by histochemical staining and immunoblotting. RESULTS: Aberrantly stained collagen was detected in 32 of 33 plaque specimens (97%) and disrupted elastic fibers in 31 of the same specimens (94%). Fibrin deposition was detected histochemically in plaque tissue from 18 of 19 patients (95%) but it was not detectable in normal or scarred tunica from control patients. The presence of authentic fibrin accumulation in plaque tissue was confirmed by immunoblot analysis but fibrin was not detected in dermal tissue extracts from the same patient. Aberrant collagen staining and fibrin deposition were detected in nodular tissue from 7 of 8 Dupuytren's contracture patients (88%) and altered elastic fibers in 5 of the same patients (63%). CONCLUSIONS: Deposition of fibrin in plaque tissue is consistent with the hypothesis that repetitive microvascular injury results in fibrin deposition in the tissue space and has served to provide insights into the pathophysiology of Peyronie's disease. We propose a model that accounts for the clinical and biological features of Peyronie's disease.

Inhibition of head and neck squamous cell carcinoma growth and invasion by the calcium influx inhibitor carboxyamido-triazole.

Local invasion and lymph node metastasis are correlated with a decreased overall survival in head and neck cancer patients and warrant new strategies to intervene in the metastatic cascade. One approach is to focus on the intracellular signaling pathways underlying the metastatic process. A common regulatory point in several signal transduction pathways is intracellular calcium homeostasis. We assessed the effect of a novel calcium influx inhibitor, carboxyamido-triazole (CAI), on the growth and invasive phenotype of cell lines derived from head and neck squamous cell carcinoma (HNSCC). CAI inhibited the growth of FaDu and EVSCC17M cells in a dose-dependent (IC50, 13-15 microM) and reversible manner. CAI also caused a generalized attenuation of receptor-mediated calcium elevation to several calcium mobilization agonists, including epidermal growth factor and bradykinin. The effects of CAI on the invasive phenotype of HNSCC cell lines were assessed by a chemo-invasion assay. HNSCC cell lines exhibited a range of invasive potential as measured by the capacity of tumor cells to penetrate a reconstituted basement membrane of Matrigel. HNSCCs were classified as highly invasive (EVSCC14M and EVSCC17M) or weakly invasive (EVSCC18, EVSCC19M, UMSCC10A, and FaDu). Treatment of HNSCC cell lines with 10 microM CAI for 24 h reduced invasion 2-14-fold in a dose-dependent manner. HNSCCs also exhibited different motilities as measured by a chemotaxis assay. EVSCC14M and EVSCC17M were highly motile, whereas EVSCC18, EVSCC19M, UMSCC10A, and FaDu were less motile. CAI reduced the migration of all cell lines. Conditioned medium from HNSCC cell lines was analyzed by zymography for production of Mr 72,000 type IV collagenase [matrix metalloproteinase (MMP)-2)] and Mr 92,000 type IV collagenase (MMP-9). All HNSCC cell lines secreted MMP-2 and/or MMP-9 into conditioned medium. Treatment of cells with 10 microM CAI for 24 h resulted in a reduction of both MMP-2 and MMP-9 production. The results demonstrate that CAI blocks cellular proliferation, migration, chemoinvasion, and MMP production by HNSCC in vitro and identify calcium-dependent signaling as a new target for inhibition of the malignant phenotype of HNSCC.

Effects of the calcium influx inhibitor carboxyamido-triazole on the proliferation and invasiveness of human prostate tumor cell lines.

Aberrant cellular signaling is a central feature of malignant cells and a potential target for anti-cancer therapy. Carboxyamido-triazole (CAI) is a calcium influx inhibitor that alters calcium-sensitive signal transduction pathways and suppresses the proliferative and metastatic potential of malignant cells. We have examined the effects of CAI on several tumor-associated parameters in human prostate cancer cell lines to evaluate the potential of CAI as a signal-transduction therapy agent for advanced-stage prostate cancer. Measuring anchorage-dependent cell growth, continuous application of CAI inhibited the growth of DU-145, PPC-1, PC3 and LNCaP tumor cells with 50% inhibitory concentrations ranging 10-30 microM. Direct cell enumeration assays revealed that the growth-suppressing activity of CAI toward DU-145 cells was reversible, indicating a cytostatic effect of the drug on tumor cells. The drug also inhibited the proliferation of several immortalized human prostatic epithelial cell lines. The proliferation of HaCaT- and RHEK-1-immortalized keratinocyte cell lines was relatively insensitive to CAI. Additionally, invasion by DU-145, PC3 and PPC-1 cells through Matrigel in vitro was reduced approximately 60-70% by 10 microM CAI. Other cellular effects of CAI included an attenuation of the elevation of intracellular free calcium in response to bombesin and carbachol in PC3 cells and a marked dose-dependent inhibition of prostate-specific antigen secretion in LNCaP cell cultures.

Amplification and expression of EMS-1 (cortactin) in head and neck squamous cell carcinoma cell lines.

Amplification of chromosome 11q13 DNA sequences is detected in approximately 30% of primary head and neck squamous cell carcinomas (HNSCC). The amplified region includes genes for cyclin D1, hst-1, int-2, and more recently, ems-1. Ems-1 encodes an 80/85kd cytoskeletal associated protein termed cortactin, which has been shown to bind F-actin and is a pp60src substrate. We investigated 16 HNSCC cell lines for ems-1 DNA amplification and gene expression by western blotting and immunofluorescence using mAb 4F11. Amplification of ems-1 DNA was detected in 8/16 (50%) cell lines and was related directly to over-expression of cortactin by western blotting and immunofluorescence. Western blotting detected both forms of cortactin, p80 and p85, at equal intensity. Immunofluorescent staining revealed low levels of cortactin localized to the cytoplasm and surface membrane in normal bronchial epithelial cells and tumor cell cultures with single copy ems-1 DNA. In contrast, tumor cell cultures with ems-1 DNA amplification demonstrated intense, homogeneous cortactin cytoplasmic staining. These results suggest that overexpression of p80/85 may be a useful marker to identify 11q13 amplification, a molecular alteration correlated with the presence of lymph node metastasis in head and neck cancer.

Frequent p53 mutations in head and neck cancer.

Squamous cell carcinomas of the head and neck (SCCHN) are associated strongly with the use of tobacco and alcohol, but little is known about the molecular pathogenesis of these tumors. In the present study, we analyzed SCCHN for mutations in the tumor suppressor gene p53 by immunocytochemistry and complementary DNA sequencing. Overexpression of p53 protein was detected in 13 (100%) of 13 SCCHN cell lines and in tumor cells cultured directly from 10 (77%) of 13 patients with SCCHN. Direct evidence for p53 mutations was obtained by sequencing p53 complementary DNA from eight SCCHN cell lines and two tumor xenografts. The genetic alterations included seven missense mutations resulting in single amino acid substitutions, a mutation encoding a stop codon, one 10-base pair deletion, and one 2-base pair addition. All seven missense mutations were G to T transversions, five of which were clustered at codons 245 and 248. A similar high frequency of G to T transversions predominates in lung cancer, another tobacco-related disease. Mutation of the p53 gene is the most common genetic alteration detected in SCCHN and implicates this gene locus as a critical site of specific damage by mutagenic carcinogens in tobacco, one of the important risk factors in the etiology of this disease.

Altered H1 histamine receptor signaling in Balb/3T3 cells transformed by v-K-ras and v-H-ras oncogenes.

Transformation of Balb/3T3 cells with the v-K-ras oncogene resulted in the expression of functional Ca(2+)-mobilizing receptors for histamine, whereas v-H-ras-transformed Balb/3T3 cells failed to show a similar response to histamine. Stimulation of histamine receptors in v-K-ras-transformed cells produced a dose-dependent increase in intracellular free calcium ([Ca2+]i), which was inhibited by the H1 histamine antagonist pyrilamine but unaffected by the H2 histamine receptor antagonist cimetidine. Histamine-mediated elevation of [Ca2+]i was partially inhibited by the removal of extracellular Ca2+, which indicates that the H1 histamine receptors mobilize intracellular Ca2+ and also promote Ca2+ influx. H1 histamine receptors were identified in both v-K-ras- and v-H-ras-transformed Balb/3T3 cells, but not in untransformed cells, using the specific H1 antagonist [3H]-pyrilamine. Transformation of Balb/3T3 cells with the viral ras oncogene results in a complex regulation of H1 histamine receptors. K-ras and H-ras transformation results in the expression of H1 histamine receptors; however, H1 receptor expression and Ca2+ mobilization are uncoupled in v-H-ras-transformed cells.

Genetic alterations in head and neck cancer.

Mutations of the ras gene family appear to be an uncommon genetic alteration in SCCHN. A common region of DNA amplification on chromosome 11q13 has been identified in SCCHN. A cluster of proto-oncogenes (int-2, hst-1, bcl-1, prad-1) has been localized to the 11q13 region. Studies are needed to determine the critical genes in 11q13 whose expression drive the amplicon. Mutations of the p53 tumor suppressor gene are the most common genetic alteration in SCCHN. The hope is that dysregulated oncogenes or tumor suppressor genes may be targets for specific therapy.

Metastatic properties of the human prostatic cell line, PPC-1, in athymic nude mice.

Limitation in the number of human prostatic cell lines has created a gap in knowledge regarding the in-vivo progression of this common cancer. The recently isolated primary prostatic carcinoma cell line, PPC-1, has been shown to be tumorigenic in athymic nude mice. These cells are now shown to form metastases to secondary sites in 10 of 12 animals in this initial study. Metastases were localized to lung and lymph nodes, and the tumor histology closely resembled that of the undifferentiated, rapidly dividing primary tumors. This is the first report describing the metastatic properties of a primary prostatic cancer cell line. PPC-1 cells are therefore likely to represent a good model system for the study of human prostate cancer progression.

Growth and histopathology of human head and neck squamous cell carcinoma implanted intraorally in nude mice.

A human squamous cell carcinoma (SCC) from the floor of the mouth (FOM) was implanted by a needle aspiration technique in the FOM site of athymic nude mice. Mice were killed at 3-week intervals, and the oral cavity, mandible, and neck were sectioned and examined histologically. Tumor growth was observed in 65% of the animals, with histologic features consistent with the engrafted human invasive SCC. These features included invasion of connective tissue in 92%, invasion of muscle in 77%, invasion and destruction of bone in 54%, and vascular invasion in 15% of the mice. In contrast, FOM tumor implanted subcutaneously on back sites of nude mice was totally encapsulated by fibrous connective tissue with evidence of capsular invasion. SCC from other head and neck sites showed similar locally invasive growth after intraoral implantation in nude mice. The results demonstrate the invasive characteristics of human head and neck SCC grown in the homologous oral cavity site in nude mice and support the nude mouse as a biologically relevant in vivo model in the investigation of the biologic characteristics and therapy of head and neck carcinoma.

Amplification of the int-2 gene in human head and neck squamous cell carcinomas.

Head and neck squamous cell carcinomas (SCC) from 21 patients were analyzed for structurally rearranged or amplified proto-oncogenes by Southern blot hybridization. The int-2 proto-oncogene was amplified 3-5 fold in 5 (50%) of 10 laryngeal SCC and 2-3 fold in 5 (45%) of 11 nonlaryngeal SCC of the head and neck. Adjacent histologically normal tissue from the same patients had single int-2 gene copy number. Coamplification of int-2 and the epidermal growth factor receptor (c-erbB-1) gene was found in one laryngeal SCC and one SCC metastatic to the neck. No amplification or structural alterations of proto-oncogenes c-erbB-2/HER2, c-myc, H-ras-1, or K-ras-2 was detected in any of the head and neck tumors. In a survey of head and neck tumor-derived cell lines, int-2 was amplified 9 fold in a hypopharyngeal tumor cell line (FaDu), but not amplified in 3 laryngeal tumor cell lines. int-2 has been localized to the q13 band of chromosome 11. We used chromosome 11 specific probes to demonstrate that int-2 amplification was not due to complete or partial chromosome 11 duplication. int-2 amplification was localized to 11q13, but did not extend to the ets-1 locus 11q23. The results indicate that int-2 is frequently amplified in SCC of the head and neck and suggest that int-2 amplification may correlate with clinical disease progression.

Beta-tubulin epitope expression in normal and malignant epithelial cells.

The expression of a unique beta-tubulin isoform (class III) was monitored in squamous cell carcinoma (SCC) and normal epithelial cells using a monoclonal tubulin antibody called TuJ1. Whole tissue homogenates of SCC, normal tissue, SCC grown in nude mice, and SCC cultured cells were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. TuJ1 antibody localization was performed using peroxidase immunostaining on paraffin sections of SCC, normal tissue, nude mouse SCC, and immunofluorescent microscopy of SCC cultured cells. The malignant tissues examined stained positive with TuJ1 and a general beta-tubulin antibody, whereas the normal tissues stained positively only for the general beta-tubulin antibody. TuJ1 epitope expression may be a useful marker for SCCs and may assist in understanding differences between normal and malignant squamous cells.

Phenotypic and cytogenetic characterization of a cell line derived from primary prostatic carcinoma.

A new cell line, PPC-I, has been established from a specimen obtained from a patient with a poorly differentiated adenocarcinoma of the prostate. This is the first line of its type derived from a primary prostatic tumor site. PPC-I cells have become immortalized in culture, exhibit transformation parameters including relaxed growth factor requirements and anchorage-independent growth, and are highly tumorigenic in nude mice. Cytogenetic studies by G-banding revealed a grossly abnormal karyotype, with a modal chromosome number of 84, multiple marker chromosomes including both homogeneously staining regions and double minutes and clonal loss of chromosomes 3, 5, 10, 15 and Y.