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.

Rapamycin induces Smad activity in prostate cancer cell lines.

Rapamycin inhibits the FK506-binding protein 12 (FKBP12)/mammalian target of rapamycin (mTOR) complex and causes cell cycle arrest in G1. The precise mechanism of growth inhibition by rapamycin is only partly understood. Rapamycin led to growth inhibition in the human prostate cancer cell lines LNCaP and PC3 cells after 72 h, ID50: 93 and 50 nM, respectively. Filter cDNA array analysis showed down-regulation by more than 0.75x by rapamycin in PC3 cells and LNCaP cells of the following genes: follistatin, eukaryotic initiation factor-4E (eIF4E), glucose-6-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH)-A, ATP synthase, heat shock protein (HSP)-1. Upregulation by more than 1.5x was found for: bone morphogenetic protein (BMP)-4, FKBP12, carcinoma embryonic antigen (CEA) precursor, eukaryotic initiation factor (eIF)-3 p36 subunit, latent transforming growth factor (TGF) beta binding protein (LTBP)1. Rapamycin induced BMP4 and reduced follistatin expression in PC3 cells. This resulted in a dose-dependent nuclear expression of Smad4 and activated the SBE4 Smad-reporter, indicating activation of TGFbeta/BMP signaling. Combining rapamycin with PI3K inhibition (LY294002) increased growth inhibition. These findings illustrate that Smad signaling plays a role in the anticancer effects of rapamycin and show that combination with PI3K inhibition improves growth inhibition.

Gene therapy of prostate cancer: current and future directions.

Prostate cancer (PCA) is the second most common cause of death from malignancy in American men. Developing new approaches for gene therapy for PCA is critical as there is no effective treatment for patients in the advanced stages of this disease. Current PCA gene therapy research strategies include cytoreductive approaches (immunotherapy and cytolytic/pro-apoptotic) and corrective approaches (replacing deleted or mutated genes). The prostate is ideal for gene therapy. It is an accessory organ, offers unique antigens (prostate-specific antigen, prostate-specific membrane antigen, human glandular kallikrein 2 etc.) and is stereotactically accessible for in situ treatments. Viral and non-viral means are being used to transfer the genetic material into tumor cells. The number of clinical trials utilizing gene therapy methods for PCA is increasing. We review the multiple issues involved in developing effective gene therapy strategies for human PCA and early clinical results.

Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.

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.

Melanin content of cultured human melanocytes and UV-induced cytotoxicity.

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.

Hypoxia-inducible factor 1alpha and 1beta proteins share common signaling pathways in human prostate cancer cells.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor consisting alpha and beta subunits. It is critically involved in cancer cell hypoxia adaptation, glycolysis, and angiogenesis. HIF-1beta is associated with HIF-1 functions as a dimerization partner of HIF-1alpha, and is on the other hand associated with carcinogenesis via dioxin signaling. Regulation of HIF-1beta protein expression was investigated in human prostate cancer (PCA) cells. HIF-1beta protein was expressed constitutively under nonhypoxic conditions in all human PCA cells tested, and was up-regulated by hypoxia, CoCl2, EGF, serum, or PMA in moderate levels. Compared to that of HIF-1alpha, the constitutive, serum-, EGF-, and PMA-increased HIF-1beta protein expression were also inhibited by selective PI3K or FRAP/TOR inhibitors but in higher doses. Hypoxia partially reversed the dose dependent inhibition of HIF-1beta. These results suggest that HIF-1alpha and beta share common signaling pathways for nuclear protein accumulation.

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.

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.

Coexpression of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor in Wilms' tumor.

BACKGROUND/PURPOSE: Hypoxia-inducible factor 1 alpha (HIF-1alpha) is an important transcriptional factor responsible for regulating expression of the angiogenic cytokine, vascular endothelial growth factor (VEGF). Little information is available regarding factors involved in the hypoxic cascade, such as HIF or VEGF in Wilms' tumor. We concomitantly evaluate the expression of HIF-1alpha and VEGF in ex vivo human Wilms' tumor specimens. METHODS: Immunohistochemical analysis (IHC) utilizing a monoclonal human anti-HIF-1alpha or a polyclonal anti-VEGF antibody was performed on ex vivo specimens of Wilms' tumor (n = 18). Predominant tumor histologic subtype was divided equally between epithelial (n = 6), blastemal (n = 6), and mixed (n = 6). Specimens were scored on a predetermined scale for distribution (percent positive cells) and intensity of HIF-1alpha/VEGF expression within areas of tumor. RESULTS: IHC analysis found that HIF-1alpha and VEGF were expressed in all Wilms' tumor specimens. Strong nuclear staining for HIF-1alpha was seen in all samples evaluated, (n = 18), mean score 2.7 (>50% cells exhibiting nuclear HIF-1alpha expression). Cytoplasmic staining for HIF-1alpha also was seen in 15 of 18 samples (83%). Distribution of VEGF was equivalent between blastemal and epthelial components, mean score 2.23 versus 2.35. CONCLUSIONS: HIF-1alpha and one of its regulatory end-products, the angiogenic cytokine VEGF, are simultaneously expressed in human Wilms' tumor. In Wilms' tumor, intratumoral hypoxia may stimulate tumor conversion to the angiogenic phenotype and incite production of VEGF. Strategies targeting the hypoxic cascade ultimately may prove efficacious against Wilms' tumor.

Coming of age: 'dysgenetics'--a theory connecting induction of persistent delayed genomic instability with disturbed cellular ageing.

INTRODUCTION: In recent years a new phenomenon has manifested itself: delayed, persistent genomic instability. When cells are treated with carcinogens not only direct induction of chromosome aberrations and mutations takes place, but there is also an indirect induction: in the distant progeny of treated cells persistently enhanced levels of new chromosome aberrations and enhanced mutation rates are found. This persistent enhanced genomic instability is not due to the presence of lesions in the DNA induced by the treatment because the response can be transmitted to untreated cells. Apparently it is caused by a persistent dysfunctioning of the cell as a whole. Due to these findings a new model for multistep carcinogenesis emerges. According to this model the initiation of carcinogenesis is the induction of a state of persistent genomic instability that not only is responsible for enhanced mutation rates of oncogenes and tumor suppressor genes, but also predisposes to immortalization. This view could lead to a radical change in our views on carcinogenesis. Therefore understanding the mechanism is of utmost importance. PURPOSE: Up to now, the mechanism responsible for this persistent delayed genomic instability remains completely elusive and has only been described as 'unknown'. In this review the phenomenon is connected with a recent theory on cellular ageing and immortalization. CONCLUSION: Although highly speculative this review provides a framework for further experimental approaches that will contribute to our understanding of delayed genomic instability and possibly even to a better understanding of cellular ageing also.

Molecular events implicated in brain tumor angiogenesis and invasion.

We have conducted studies designed to help elucidate the molecular mechanisms involved in brain tumor invasion and angiogenesis, which are critical in the growth of malignant tumors of the central nervous system. A variety of molecular factors have been implicated in these processes. Here we focus on three that are of particular importance in the progression of brain tumors. Angiopoietins are involved in the regulation of vascular development. Hypoxia inducible factor-1 is a transcription factor that up-regulates genes, including genes encoding vascular endothelial growth factor under hypoxic conditions. Focal adhesion kinase is associated with infiltration of tumor cells and angiogenesis.

Cancer cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer as vaccines for the treatment of genitourinary malignancies.

When irradiated and administered intradermally as vaccines, cancer cells engineered to secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) by gene transfer elicit potent anticancer immune responses in a variety of animal tumor models. Upon vaccination, antigens present in the cancer cells are phagocytosed and processed by skin dendritic cells. These dendritic cells then prime anticancer immune responses by presenting antigenic peptides to T cells. The immune responses generated are capable of eradicating small but lethal cancer cell inocula with minimal toxicity in preclinical animal tumor studies. To develop this vaccination strategy for the treatment of human genitourinary cancers, we have conducted phase I clinical trials using human genitourinary cancer cells as sources of cancer cell antigens. In the first human clinical trial of genetically engineered cancer cell vaccines, a phase I clinical trial of kidney cancer cell vaccines (n = 18), kidney cancer cells were removed at surgery, propagated briefly in vitro, and then genetically modified to secrete high levels of GM-CSF via ex vivo transduction with the retrovirus MFG-GM-CSF. After irradiation, the kidney cancer cells were administered as vaccines to 18 patients with advanced kidney cancers. Vaccine treatment, which caused few side effects, nonetheless appeared to trigger anticancer immune responses manifest as conversion of delayed-type hypersensitivity (DTH) skin responses against irradiated autologous cancer cells after vaccination. Biopsies of vaccine sites yielded findings reminiscent of biopsies from preclinical animal model studies, with evidence of vaccine cell recruitment of dendritic cells, T cells, and eosinophils. One patient with measurable kidney cancer metastases treated at the highest vaccine dose level experienced a partial treatment response. The bioactivity of GM-CSF-secreting autologous cancer cell vaccines was confirmed in a phase I clinical trial for prostate cancer (n = 8). Vaccine cells were prepared from surgically harvested prostate tumors by ex vivo transduction with MFG-GM-CSF in a manner similar to that used for the kidney cancer trial. Vaccine treatment was well tolerated and associated with induction of anticancer immunity as assessed using DTH skin testing. In addition, new antiprostate cancer cell antibodies were detected in serum samples from treated men as a consequence of vaccination. These first clinical trials of GM-CSF-secreting cancer cell vaccines for the treatment of genitourinary cancers have demonstrated both safety and bioactivity, in that very few side effects have been seen and anticancer immune responses have been detected. Future clinical studies will be required to assess vaccine treatment efficacy, refine vaccination dose and schedule, define the appropriate clinical context for the use of such vaccines, and ascertain optimal combinations involving vaccines and other local or systemic anticancer treatments.

Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression.

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) plays a critical role in angiogenesis during vascular development. The authors tested the hypothesis that HIF-1 expression correlates with progression and angiogenesis in brain tumors. METHODS: The authors investigated the expression of the HIF-1alpha and HIF-1beta subunits in human glioma cell lines and brain tumor tissues using Western blot analysis and immunohistochemistry. RESULTS: In glioblastomas multiforme (GBMs), HIF-1alpha primarily was localized in pseudopalisading cells around areas of necrosis and in tumor cells infiltrating the brain at the tumor margin. In contrast, HIF-1alpha was expressed in stromal cells throughout hemangioblastomas (HBs). Like HIF-1alpha, HIF-1beta was most highly expressed in high grade tumors but was expressed more widely than HIF-1alpha, including cells away from necrotic zones. In the brains of mice injected with Glioma 261 cells, a pattern of HIF-1alpha expression identical to that observed in human GBMs was noted. CONCLUSIONS: In GBMs, the heterogeneous pattern of HIF-1alpha expression appears to be determined at least in part by tissue oxygenation, whereas in HBs the homogeneous expression of HIF-1alpha may be driven by an oncogenic rather than a physiologic stimulus.

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.

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.

Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer.

Vaccination with irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting gene-transduced cancer vaccines induces tumoricidal immune responses. In a Phase I human gene therapy trial, eight immunocompetent prostate cancer (PCA) patients were treated with autologous, GM-CSF-secreting, irradiated tumor vaccines prepared from ex vivo retroviral transduction of surgically harvested cells. Expansion of primary cultures of autologous vaccine cells was successful to meet trial specifications in 8 of 11 cases (73%); the yields of the primary culture cell limited the number of courses of vaccination. Side effects were pruritus, erythema, and swelling at vaccination sites. Vaccine site biopsies manifested infiltrates of dendritic cells and macrophages among prostate tumor vaccine cells. Vaccination activated new T-cell and B-cell immune responses against PCA antigens. T-cell responses, evaluated by assessing delayed-type hypersensitivity (DTH) reactions against untransduced autologous tumor cells, were evident in two of eight patients before vaccination and in seven of eight patients after treatment. Reactive DTH site biopsies manifested infiltrates of effector cells consisting of CD45RO+ T-cells, and degranulating eosinophils consistent with activation of both Th1 and Th2 T-cell responses. A distinctive eosinophilic vasculitis was evident near autologous tumor cells at vaccine sites, and at DTH sites. B-cell responses were also induced. Sera from three of eight vaccinated men contained new antibodies recognizing polypeptides of 26, 31, and 150 kDa in protein extracts from prostate cells. The 150-kDa polypeptide was expressed by LNCaP and PC-3 PCA cells, as well as by normal prostate epithelial cells, but not by prostate stromal cells. No antibodies against prostate-specific antigen were detected. These data suggest that both T-cell and B-cell immune responses to human PCA can be generated by treatment with irradiated, GM-CSF gene-transduced PCA vaccines.

Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia.

The appropriate choice of an internal standard is critical for quantitative RNA analyses. As housekeeping genes, GAPDH, beta-actin, cyclophilin, and 28S rRNA are commonly employed as RNA internal standards with the assumption that their expression levels remain relatively constant in different experimental conditions. We tested this assumption under hypoxia (1% O2, 24 hours) compared to normoxia (20% O2, 24 hours) and compared RNA levels of these 4 housekeeping genes head to head using ribonuclease protection assays. Four biologically diverse cell lines with respect to clonal origin, neoplastic transformation, and growth rates were used in the comparison. Expression levels of 28S rRNA were constant, independent of O2 tension, but levels of GAPDH, beta-actin, and cyclophilin varied widely with hypoxia. In particular, GAPDH mRNA expression was increased by 21.2-75.1% under hypoxic conditions. Increased GAPDH transcription in hypoxia was correlated in the cancer cell lines with upregulation of the transcription factor Hypoxia Inducible Factor-1alpha protein levels in identical experimental conditions. These results suggest that 28S rRNA is a reliable internal control for comparative analyses of transcription under hypoxia; GAPDH appears particularly unfavorable for this purpose either in hypoxia or other experimental conditions that upregulate HIF-1alpha.