Identification of endothelin-1 in the pathophysiology of metastatic adenocarcinoma of the prostate.

Prostate cancer is the second most common cause of death from cancer in U.S. men, and advanced, hormone-refractory disease is characterized by painful osteoblastic bone metastases. Endothelin-1, more commonly known as a potent vasoconstrictor, is a normal ejaculate protein that also stimulates osteoblasts. We show here that plasma immunoreactive endothelin concentrations are significantly elevated in men with metastatic prostate cancer and that every human prostate cancer cell line tested produces endothelin-1 messenger RNA and secretes immunoreactive endothelin. Exogenous endothelin-1 is a prostate cancer mitogen in vitro and increases alkaline phosphatase activity in new bone formation, indicating that ectopic endothelin-1 may be a mediator of the osteoblastic response of bone to metastatic prostate cancer.

Cyclosporine A, an in vitro calmodulin antagonist, induces nuclear lobulations in human T cell lymphocytes and monocytes.

Cyclosporine A is a noncytotoxic, natural, 11 amino acid cyclic peptide used clinically as an immunosuppressant to prevent organ rejection after transplantation. Cyclosporine A is an in vitro calmodulin antagonist. At the low concentrations required to inhibit calmodulin-dependent phosphodiesterase in vitro, cyclosporine A causes a dramatic alteration in the nuclear morphology of 23% of human peripheral blood mononuclear leukocytes in vitro without loss of viability. The shape of the nucleus changes from ovoid to a distinctive, radially splayed lobulated structure. The changes occur in a dose-dependent manner in 60 min at 37 degrees C. Specific monoclonal antibodies to human leukocytes identify the cells susceptible to nuclear lobulation by cyclosporine A as OKT4 antigen-positive T cell lymphocytes and monocytes. The lobulated nuclei are 2N as determined by flow cytometric measurement of ethidium bromide fluorescence of DNA. The cyclosporine A-induced lobulation of T cell nuclei requires both physiologic temperature and metabolic energy. Although structurally different than cyclosporine A, the calmodulin antagonists R24571 and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide] also produce T cell nuclear lobulations that are indistinguishable from the nuclear lobulations caused by cyclosporine A. These data indicate that nonmitotic structural elements that govern normal nuclear morphology in a subset of mononuclear leukocytes appear to require a calmodulin-mediated process. Cyclosporine A may be a useful noncytotoxic inhibitor of calmodulin-dependent systems that influence nuclear structure and function.

Identification of a chromosome 18q gene that is altered in colorectal cancers.

Allelic deletions involving chromosome 18q occur in more than 70 percent of colorectal cancers. Such deletions are thought to signal the existence of a tumor suppressor gene in the affected region, but until now a candidate suppressor gene on this chromosomal arm had not been identified. A contiguous stretch of DNA comprising 370 kilobase pairs (kb) has now been cloned from a region of chromosome 18q suspected to reside near this gene. Potential exons in the 370-kb region were defined by human-rodent sequence identities, and the expression of potential exons was assessed by an "exon-connection" strategy based on the polymerase chain reaction. Expressed exons were used as probes for cDNA screening to obtain clones that encoded a portion of a gene termed DCC; this cDNA was encoded by at least eight exons within the 370-kb genomic region. The predicted amino acid sequence of the cDNA specified a protein with sequence similarity to neural cell adhesion molecules and other related cell surface glycoproteins. While the DCC gene was expressed in most normal tissues, including colonic mucosa, its expression was greatly reduced or absent in most colorectal carcinomas tested. Somatic mutations within the DCC gene observed in colorectal cancers included a homozygous deletion of the 5' end of the gene, a point mutation within one of the introns, and ten examples of DNA insertions within a 0.17-kb fragment immediately downstream of one of the exons. The DCC gene may play a role in the pathogenesis of human colorectal neoplasia, perhaps through alteration of the normal cell-cell interactions controlling growth.

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

Hypoxia-inducible factor-1 in human breast and prostate cancer.

The tumor microenvironment is best characterized as a fluctuation of hypoxia and nutrient deprivation, which leads to epigenetic and genetic adaptation of clones and increased invasiveness and metastasis. In turn, these hypoxic adaptations make the tumors more difficult to treat and confer increased resistance to current therapies. Part of this adaptation is the regulation of gene products in response to hypoxia. Many of these hypoxia-regulated genes are mediated by the hypoxia-inducible factor 1 (HIF-1) complex, which is composed of a heterodimer pair of HIF-1alpha and HIF-1beta. This heterodimer binds to the promoter of hypoxia-responsive genes, while interacting with other transcription factors, such as p300, signal and transducer of transcription 3, and Redox effector factor 1/apurinic/apyrimidinic endonuclease. HIF-1alpha levels itself can be regulated by hypoxia transcriptionally and post-translationally through ubiquitination; but the magnitude of the response is modulated by several other pathways, including free radicals that affect crosstalk with HIF-1alpha/HIF-1beta transcriptional activities. HIF-1alpha has emerged as an important transcription factor in breast cancer and prostate cancer biology, and is expressed in the early stages of mammary and prostate carcinogenesis. Its expression is correlated with diagnostic and prognostic indicators for early relapse and metastatic disease, thus making HIF-1alpha a potential prognostic biomarker in proteomic assessments of breast and prostate cancers. The importance of HIF-1alpha in tumor progression makes it a logical target for chemoprevention strategies in patients at higher genetic risk of breast and prostate cancer with Cox 2 inhibitors or 2-methoxyestradiol, as well as a target for new approaches to inhibiting angiogenesis. The crosstalk between estrogen signaling pathways and HIF-1alpha is still not fully defined in breast cancer, but downstream estrogen receptor signaling may be a candidate for estrogen modulation of HIF-1alpha levels. In prostate cancer, androgens upregulate HIF-1alpha through androgen-regulated autocrine receptor tyrosine kinase receptor signaling. This review will put into perspective the role of HIF-1alpha in endocrine oncology and present new data on HIF-1alpha signaling and the potential for targeted therapies, including combinatory hormonal therapies.

Molecular characterization of defective antigen processing in human prostate cancer.

BACKGROUND: Gene-modified tumor cell vaccines have shown efficacy in animal models of malignancy, including prostate cancer. Class I major histocompatibility complex (MHC) assembly and function in the cellular targets of such therapies is pivotal in determining the efficacy of specific cytokine-secreting tumor vaccines. PURPOSE: To help guide development of genetically engineered vaccine therapy for human prostate cancer, potential immune resistance pathways were evaluated by analysis of class I MHC assembly in prostate cancer cells. METHOD: Class I MHC assembly in metastasis-derived human prostate cancer cell lines (LNCaP, PPC-1, DU-145, PC-3, and TSU) and a normal prostate-derived cell line (TP-2) were characterized by phenotypic, molecular, and functional assays. Assembled class I MHC and antigen was measured by flow cytometry; mRNA levels of assembly components (class I MHC heavy chain, beta 2-microglobulin, and the antigen transporter gene product TAP-2) were determined; and antigen processing was measured with a chimeric reconstituted system using vaccinia vectors. Restoration of antigen processing was attempted by interferon gamma stimulation and by transfection with mouse class I MHC heavy-chain cDNA. RESULTS: Assembled class I MHC was underexpressed in two (LNCaP and PPC-1) of five prostate cancer cell lines compared with normal prostate-derived controls. PPC-1 cells underexpressed TAP-2 mRNA despite abundant class I MHC and beta 2-microglobulin message. Induction of TAP-2 by interferon gamma indicated that coding sequences for TAP-2 message were present in PPC-1. Resistance to cytotoxic T lymphocytes (CTL) lysis showed a functional defect in antigen transport by PPC-1 cells; reversal of the molecular defect with interferon gamma led to restoration of functional antigen processing. In contrast, LNCaP cells had competent antigen transport but deficient class I MHC heavy-chain function despite abundant class I MHC RNA; though refractory to stimulation by interferon gamma, this defect responded to transfection of class I MHC heavy-chain cDNA. CONCLUSIONS: Metastatic prostate cancer cells can escape T-cell recognition via divergent mechanisms of defective class I MHC assembly. The specific underexpression of TAP-2 gene product in PPC-1 cells contrasts with prior studies of TAP gene underexpression in lung cancer (which concurrently underexpressed class I MHC heavy chain) and provides evidence for a regulatory pathway controlling TAP-2 gene expression in human cancers that may not affect class I MHC heavy-chain expression. IMPLICATIONS: In clinical application of gene therapy for prostate cancer, these findings provide a rationale for focusing on strategies that can circumvent sole reliance on class I MHC-mediated tumor cell recognition by CTL.

Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates gene expression in critical pathways involved in tumor growth and metastases. In this report, we investigated whether the level of HIF-1 alpha is increased during carcinogenesis in breast tissue and is associated with other tumor biomarkers. METHODS: Paraffin-embedded clinical specimens from five pathologic stages of breast tumorigenesis and from normal breast tissue were used. HIF-1 alpha protein and the biomarkers vascular endothelial growth factor (VEGF), HER-2/neu, p53, Ki-67, and estrogen receptor (ER) were identified immunohistochemically, and microvessel density (a measure of angiogenesis) was determined. Associations among levels of HIF-1 alpha and these biomarkers were tested statistically. All statistical tests are two-sided. RESULTS: The frequency of HIF-1 alpha-positive cells in a specimen increased with the specimen's pathologic stage (P<.001, chi(2) test for trend) as follows: normal breast tissue (0 specimens with > or = 1% HIF-1 alpha-positive cells in 10 specimens tested), ductal hyperplastic lesions (0 in 10), well-differentiated ductal carcinomas in situ (DCIS) (11 in 20), well-differentiated invasive breast cancers (12 in 20), poorly differentiated DCIS (17 in 20), and poorly differentiated invasive carcinomas (20 in 20). Increased levels of HIF-1 alpha were statistically significantly associated with high proliferation and increased expression of VEGF and ER proteins. In DCIS lesions, increased levels of HIF-1 alpha were statistically significantly associated with increased microvessel density. HIF-1alpha showed a borderline association with HER-2/neu but no association with p53. CONCLUSIONS: The level of HIF-1 alpha increases as the pathologic stage increases and is higher in poorly differentiated lesions than in the corresponding type of well-differentiated lesions. Increased levels of HIF-1 alpha are associated with increased proliferation and increased expression of ER and VEGF. Thus, increased levels of HIF-1 alpha are potentially associated with more aggressive tumors.

Immortalization of Syrian hamster embryo cells is in itself a multistep event.

The hypothesis that induction of immortalization of rodent cells follows one-hit kinetics was tested by the determination of frequencies of immortalization of Syrian hamster embryo cells after treatment with benzo(a)pyrene, X-rays, or ethylnitrosourea. Contrary to expectation, immortalization did not occur in a single step. Full immortalization appeared to be a process which required at least three steps: extension of life span (step 1), increase in cloning efficiency (step 2), and increase in growth rate (step 3). These three steps occur in this fixed sequence. The first step appears to be induced by the carcinogenic treatment, while the two other steps occur spontaneously in the progeny of cells which underwent the first step. The frequency of induction of the first step is in the order of magnitude of mutation induction, which suggests that mutation in one allele of a limited number of loci is sufficient to initiate the process of immortalization. However, the spontaneous frequency of immortalization is below 2.4 x 10(-9)/cell/generation, which appears to be too low for a spontaneous mutation frequency. The frequencies/cell/generation of the second and the third step are in the order of magnitude of spontaneous mutation frequencies.

Immortalization of carcinogen-treated Syrian hamster embryo cells occurs indirectly via an induced process.

The hypothesis that rodent cells can be immortalized by the direct induction of a single mutation-like event was tested by initiating cultures of benzo(a)pyrene treated Syrian hamster embryo cells with low inocula and expanding these few cells maximally until senescence prevented further culturing or immortalization took place. According to the mutation hypothesis immortalization is hardly to be expected under these conditions. However, immortalization was frequently observed. Therefore the induction of immortalization appears indirect. The progeny of benzo(a)pyrene treated cells immortalized with a rate of 3.9 x 10(-8)/cell/generation, which is 64 times higher than the spontaneous rate. The results are in line with the probabilistic theory developed in 1980 by both Fernandez et al. (Proc. Natl. Acad. Sci. USA, 77: 7272-7276, 1980) and Kennedy et al. (Proc. Natl. Acad. Sci. USA, 77: 7262-7266, 1980), which states that treatment of cells with a carcinogen can result in a so-called activated state of the treated cells which is transmitted to the progeny and which results in an enhanced rate of transforming events.

Immortalization of Syrian hamster embryo cells: probabilistic event or deterministic process.

Previous findings on the induction of immortalization in SHE cells have been explained with the activation/alteration hypothesis which postulates that treatment with a carcinogen results in the induction of a so-called "activated state" which enhances the rate of a probabilistic event in the progeny of the treated cells. This event is supposed to be a mutation. Because it has been recently indicated that in mammalian cells the switching on of signal transduction pathways by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or carcinogens can lead to genetic instability in the progeny of the treated cells, the possibility of an analogy between the induction of genetic instability and induction of immortalization after treatment with TPA was investigated. No effect of TPA was found on the rate of immortalization/cell/generation, not in otherwise untreated cells nor in cells treated with benzo(a)pyrene. TPA was found to enhance the life span of SHE cells. The life span of a culture correlated with its growth rate and its cell density at confluence both in the absence and presence of TPA. These correlations are supposed to reflect a regulation mechanism involved in the program of cellular senescence, and supposedly TPA can partly reverse this program. Treatment with benzo(a)pyrene also interferes with the life span resulting in premature senescence in most of the cells and extension of life span in a small fraction of the cells which subsequently can become immortal. Repeated switching from logarithmic growth to G0 also enhanced life span and rate of immortalization. The findings indicate that the activated state is a disturbance of a differentiation program affecting in SHE cells the program of cellular senescence and that, as an explanation for immortalization, epigenetic alterations causing a deterministic process of dedifferentiation in a subpopulation of the cells appear as plausible or perhaps even more plausible as a probabilistic mutation. This indicates that disturbance of differentiation might be among the causes of genetic instability.

Ataxia-telangiectasia-like Chinese hamster V79 cell mutants with radioresistant DNA synthesis, chromosomal instability, and normal DNA strand break repair.

We have isolated three radiosensitive mutants (V-C4, V-E5, and V-G8) of the Chinese hamster V79 cell line which also show increased sensitivities to killing by bleomycin (approximately 2-5-fold) and ethyl methanesulfonate (approximately 2-fold). Genetic complementation analysis indicates that all three mutants belong to one complementation group. The mutants show a radioresistant DNA synthesis following X-ray irradiation when compared to wild-type V79 cells. Both the level and the rate of repair of DNA single- and double-strand breaks measured by DNA elution were similar to those observed in wild-type V79 cells. The level of spontaneously occurring chromosome aberrations in two of these mutants differs severalfold from the level observed in wild-type V-79 cells and in V-G8, to approximately 2- and 6-fold increase in V-E5 and V-C4, respectively. X-irradiation of the mutants resulted in consistently 3-4-fold higher levels of chromatid gaps, breaks, and exchanges than observed in wild-type V79 cells. In addition, G1 irradiation of the mutant cells yielded both chromosome and chromatid types of aberrations. The level and pattern of chromosomal aberrations induced by X-rays in V-C4, V-E5, and V-G8 are similar to those observed in ataxia-telangiectasia cells. These results indicate that our mutants represent the first rodent cell mutants which show phenotypic characteristics strongly resembling those in cells from ataxia-telangiectasia patients.

Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells.

Prostate-specific antigen (PSA) is a widely used marker for the diagnosis and management of prostate cancer. Minimal enhancer/promoter constructs derived from the 5' flank of the human PSA gene (prostate-specific enhancer) were inserted into adenovirus type 5 DNA so as to drive the E1A gene, thereby creating a prostate-specific enhancer-containing virus, CN706. E1A was expressed at high levels in CN706-infected human PSA-producing LNCaP cells but not in CN706-infected DU145 cells, which are human prostate cells that do not express PSA. The titer of CN706 was significantly higher in LNCaP cells compared to several human cell lines that do not produce PSA (HBL100, PANC-1, MCF-7, DU145, and OVCAR3). Furthermore, in LNCaP cells, the yield of CN706 was dependent on exogenous androgen (R1881). CN706 destroyed large LNCaP tumors (1 x 10(9) cells) and abolished PSA production in nu/nu mouse xenograft models with a single intratumoral injection.

Angiogenesis and prostate cancer: identification of a molecular progression switch.

To elucidate the sequence of molecular events intricate with angiogenesis and the initiation and progression prostate cancer, the temporal and spatial expression patterns of platelet endothelial cell adhesion molecule-1 (PECAM1/CD31), hypoxia-induced factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and the cognate receptors VEGFR1 and VEGFR2 were characterized. Immunohistochemical and in situ analyses of prostate tissue specimens derived from the spontaneous autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) model identified a distinct early angiogenic switch consistent with the expression of PECAM-1, HIF-1alpha, and VEGFR1 and the recruitment of new vasculature to lesions representative of high-grade prostatic epithelial neoplasia (PIN). During progression of prostate cancer, the intraductal microvessel density (IMVD) was also observed to increase as a function of tumor grade. Immunoblot and in situ analyses further demonstrated a distinct late angiogenic switch consistent with decreased expression of VEGFR1, increased expression of VEGFR2, and the transition from a differentiated adenocarcinoma to a more poorly differentiated state. Analysis of clinical prostate cancer specimens validated the predictions of the TRAMP model. This resolution of prostate cancer-associated angiogenesis into distinct early and late molecular events establishes the basis for a "progression-switch" model to explain how the targets of antiangiogenic therapy might change as a function of tumor progression.

Evidence supporting exclusion of the DCC gene and a portion of chromosome 18q as the locus for susceptibility to hereditary nonpolyposis colorectal carcinoma in five kindreds.

Hereditary non-polyposis colorectal carcinoma (HNPCC) syndrome is characterized by early onset and multiple cancers of predominantly the proximal colon and occasionally other organs. The mode of transmission is compatible with autosomal dominant inheritance but the location and characteristics of the putative susceptibility gene are unknown. We performed linkage analyses with the aim of proving or excluding the existence of a susceptibility locus on 18q. This hypothesis was based on the frequent involvement of the DCC gene in colorectal carcinoma and on the previously reported linkage between HNPCC and the Kidd blood group locus (JK) also on 18q. Seven HNPCC families were tested with eight polymorphisms, including three from within DCC. The DCC locus could be excluded as the HNPCC susceptibility locus in five families in which the two point logarithm-of-odds scores were -3.66, -3.63, -4.12, -7.90, and -3.74 at the recombination fraction of 0.00. In the remaining two families linkage could be neither excluded nor confirmed. The added pairwise logarithm-of-odds score for all seven families was -22.65 at the recombination fraction of 0.00. Multipoint analyses of linkage in the seven families suggested exclusion of some 60 cM in the region DCC-D18S18-D18S22-D18S7 as the site for HNPCC susceptibility locus. In addition to DCC, the excluded portion comprises JK.

Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.

Dysregulated signal transduction from receptor tyrosine kinases to phosphatidylinositol 3-kinase (PI3K), AKT (protein kinase B), and its effector FKBP-rapamycin-associated protein (FRAP) occurs via autocrine stimulation or inactivation of the tumor suppressor PTEN in many cancers. Here we demonstrate that in human prostate cancer cells, basal-, growth factor-, and mitogen-induced expression of hypoxia-inducible factor 1 (HIF-1) alpha, the regulated subunit of the transcription factor HIF-1, is blocked by LY294002 and rapamycin, inhibitors of PI3K and FRAP, respectively. HIF-1-dependent gene transcription is blocked by dominant-negative AKT or PI3K and by wild-type PTEN, whereas transcription is stimulated by constitutively active AKT or dominant-negative PTEN. LY294002 and rapamycin also inhibit growth factor- and mitogen-induced secretion of vascular endothelial growth factor, the product of a known HIF-1 target gene, thus linking the PI3K/PTEN/AKT/FRAP pathway, HIF-1, and tumor angiogenesis. These data indicate that pharmacological agents that target PI3K, AKT, or FRAP in tumor cells inhibit HIF-1alpha expression and that such inhibition may contribute to therapeutic efficacy.

Increased expression of hypoxia inducible factor-1alpha in rat and human prostate cancer.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates genes involved in adaptation to hypoxia. Expression of HIF-1alpha was evaluated in rat and human prostate cancer cell lines. Increased expression of HIF-1alpha mRNA in rat prostate cancer cell lines and hypoxia-induced expression of HIF-1alpha protein in human prostate cancer cell lines are associated with increased cell growth rates and metastatic potential. HIF-1alpha mRNA was undetectable in the normal rat ventral prostate by Northern blot hybridization. HIF-1alpha protein expression and HIF-1 DNA binding activity were detected in normoxic PC-3 cells. Human prostate cancer cells plated at low density manifested higher functional HIF-1alpha expression than cells plated at high density independent of O2 tension. HIF-1alpha may become dysregulated in prostate cancer and thus drive the transcription of hypoxia-adaptive genes involved in tumor progression. This is also the first evidence that human cancer cells can express functional HIF-1alpha protein under normoxic conditions.

Endothelin-1 production and decreased endothelin B receptor expression in advanced prostate cancer.

The potent vasoconstrictor endothelin-1 (ET-1) is at its highest concentration in the normal human ejaculate and is associated with the progression of metastatic prostate cancer. ET-1 protein expression is detected in situ in 14 of 14 primary cancers and 14 of 16 metastatic sites of human prostatic carcinoma. Exogenous ET-1 induces prostate cancer proliferation directly and enhances the mitogenic effects of insulin-like growth factor I, insulin-like growth factor II, platelet-derived growth factor, basic fibroblast growth factor, and epidermal growth factor in serum-free conditions in vitro. The ETA-selective receptor antagonist A-127722 inhibits ET-1-stimulated growth, but the ETB-selective receptor antagonist BQ-788 does not. ET-3, an ETB-selective agonist, also had no effect on prostate cancer growth. No specific ETB-binding sites could be demonstrated in any established human prostate cancer cell line tested, and ETB mRNA, detected by reverse transcription PCR, was reduced. The predominance of ETB binding on human benign prostatic epithelial tissue is not present in metastatic prostate cancer by autoradiography. In human prostate cancer progression to metastases, ET-1 and ETA expression are retained, whereas ETB receptor expression is reduced.

Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases.

Neovascularization and increased glycolysis, two universal characteristics of solid tumors, represent adaptations to a hypoxic microenvironment that are correlated with tumor invasion, metastasis, and lethality. Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. HIF-1 transcriptional activity is determined by regulated expression of the HIF-1alpha subunit. In this study, HIF-1alpha expression was analyzed by immunohistochemistry in 179 tumor specimens. HIF-1alpha was overexpressed in 13 of 19 tumor types compared with the respective normal tissues, including colon, breast, gastric, lung, skin, ovarian, pancreatic, prostate, and renal carcinomas. HIF-1alpha expression was correlated with aberrant p53 accumulation and cell proliferation. Preneoplastic lesions in breast, colon, and prostate overexpressed HIF-1alpha, whereas benign tumors in breast and uterus did not. HIF-1alpha overexpression was detected in only 29% of primary breast cancers but in 69% of breast cancer metastases. In brain tumors, HIF-1alpha immunohistochemistry demarcated areas of angiogenesis. These results provide the first clinical data indicating that HIF-1alpha may play an important role in human cancer progression.

A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy.

CV706 is a prostate-specific antigen (PSA)-selective, replication-competent adenovirus that has been shown to selectively kill human prostate cancer xenografts in preclinical models. To study the safety and activity of intraprostatic delivery of CV706, a Phase I dose-ranging study for the treatment of patients with locally recurrent prostate cancer after radiation therapy was conducted. Twenty patients in five groups were treated with between 1 x 10(11) and 1 x 10(13) viral particles delivered by a real-time, transrectal ultrasound-guided transperineal technique using a three-dimensional plan. The primary end point was the determination of treatment-related toxicity. Secondary objectives included evaluation of the antitumor activity of CV706 and monitoring for other correlates of antineoplastic action. In this study, CV706 was found to be safe and was not associated with irreversible grade 3 or any grade 4 toxicity. No grade >1 alterations in liver function tests associated with CV706 administration were observed. Posttreatment prostatic biopsies and detection of a delayed "peak" of circulating copies of virus provided evidence of intraprostatic replication of CV706. The study defined the timing of CV706 shedding into blood and urine as well as the appearance of circulating Ad5 neutralizing antibodies. Finally, this study documents the serum PSA response of treated patients and reveals a dose response showing that all five patients who achieved a > or =50% reduction in PSA were treated with the highest two doses of CV706. This study represents the first clinical translation of a prostate-specific, replication-restricted adenovirus for the treatment of prostate cancer. Taken together, this study documents that intraprostatic delivery of CV706 can be safely administered to patients, even at high doses, and the data also suggest that CV706 possesses enough clinical activity, as reflected by changes in serum PSA, to warrant additional clinical and laboratory investigation.

Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced, irradiated tumor vaccines induce potent, T-cell-mediated antitumor immune responses in preclinical models. We report the initial results of a Phase I trial evaluating this strategy for safety and the induction of immune responses in patients with metastatic renal cell carcinoma (RCC). Patients were treated in a randomized, double-blind dose-escalation study with equivalent doses of autologous, irradiated RCC vaccine cells with or without ex vivo human GM-CSF gene transfer. The replication-defective retroviral vector MFG was used for GM-CSF gene transfer. No dose-limiting toxicities were encountered in 16 fully evaluable patients. GM-CSF gene-transduced vaccines were equivalent in toxicity to nontransduced vaccines up to the feasible limits of autologous tumor vaccine yield. No evidence of autoimmune disease was observed. Biopsies of intradermal sites of injection with GM-CSF gene-transduced vaccines contained distinctive macrophage, dendritic cell, eosinophil, neutrophil, and T-cell infiltrates similar to those observed in preclinical models of efficacy. Histological analysis of delayed-type hypersensitivity responses in patients vaccinated with GM-CSF-transduced vaccines demonstrated an intense eosinophil infiltrate that was not observed in patients who received nontransduced vaccines. An objective partial response was observed in a patient treated with GM-CSF gene-transduced vaccine who displayed the largest delayed-type hypersensitivity conversion. No replication-competent retrovirus was detected in vaccinated patients. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous GM-CSF gene-transduced tumor vaccine for RCC patients.