Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men.

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the ß-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between ß-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2'-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.

Identification of novel DNA-methylated genes that correlate with human prostate cancer and high-grade prostatic intraepithelial neoplasia.

BACKGROUND: Prostate cancer (PCa) harbors a myriad of genomic and epigenetic defects. Cytosine methylation of CpG-rich promoter DNA is an important mechanism of epigenetic gene inactivation in PCa. There is considerable amount of data to suggest that DNA methylation-based biomarkers may be useful for the early detection and diagnosis of PCa. In addition, candidate gene-based studies have shown an association between specific gene methylation and alterations and clinicopathologic indicators of poor prognosis in PCa. METHODS: To more comprehensively identify DNA methylation alterations in PCa initiation and progression, we examined the methylation status of 485 577 CpG sites from regions with a broad spectrum of CpG densities, interrogating both gene-associated and non-associated regions using the recently developed Illumina 450K methylation platform. RESULTS: In all, we selected 33 promoter-associated novel CpG sites that were differentially methylated in high-grade prostatic intraepithelial neoplasia and PCa in comparison with benign prostate tissue samples (false discovery rate-adjusted P-value <0.05; ß-value 0.2; fold change >1.5). Of the 33 genes, hierarchical clustering analysis demonstrated BNC1, FZD1, RPL39L, SYN2, LMX1B, CXXC5, ZNF783 and CYB5R2 as top candidate novel genes that are frequently methylated and whose methylation was associated with inactivation of gene expression in PCa cell lines. Pathway analysis of the genes with altered methylation patterns identified the involvement of a cancer-related network of genes whose activity may be regulated by TP53, MYC, TNF, IL1 and 6, IFN-γ and FOS in prostate pathogenesis. CONCLUSION: Our genome-wide methylation profile shows epigenetic dysregulation of important regulatory signals in prostate carcinogenesis.

A common nonsense mutation in EphB2 is associated with prostate cancer risk in African American men with a positive family history.

BACKGROUND: The EphB2 gene was recently implicated as a prostate cancer (PC) tumour suppressor gene, with somatic inactivating mutations occurring in approximately 10% of sporadic tumours. We evaluated the contribution of EphB2 to inherited PC susceptibility in African Americans (AA) by screening the gene for germline polymorphisms. METHODS: Direct sequencing of the coding region of EphB2 was performed on 72 probands from the African American Hereditary Prostate Cancer Study (AAHPC). A case-control association analysis was then carried out using the AAHPC probands and an additional 183 cases of sporadic PC compared with 329 healthy AA male controls. In addition, we performed an ancestry adjusted association study where we adjusted for individual ancestry among all subjects, in order to rule out a spurious association due to population stratification. RESULTS: Ten coding sequence variants were identified, including the K1019X (3055A-->T) nonsense mutation which was present in 15.3% of the AAHPC probands but only 1.7% of 231 European American (EA) control samples. We observed that the 3055A-->T mutation significantly increased risk for prostate cancer over twofold (Fisher's two sided test, p = 0.003). The T allele was significantly more common among AAHPC probands (15.3%) than among healthy AA male controls (5.2%) (odds ratio 3.31; 95% confidence interval 1.5 to 7.4; p = 0.008). The ancestry adjusted analyses confirmed the association. CONCLUSIONS: Our data show that the K1019X mutation in the EphB2 gene differs in frequency between AA and EA, is associated with increased risk for PC in AA men with a positive family history, and may be an important genetic risk factor for prostate cancer in AA.

Activated eosinophils upregulate the metastasis suppressor molecule E-cadherin on prostate tumor cells.

Cell adhesion molecules (CAMs) play an important role in cancer metastasis by facilitating attachment to vascular endothelia, invasion and spread into secondary tissue sites. We have shown that activated eosinophils (EosA) inhibited the growth of prostate cancer (Pca) cells in vitro. In the present study, we examined the ability of EosA 24 hr conditioned supernatants (EosAcs) to modulate the expression of ICAM-1, VCAM-1, ELAM-1, E-cadherin and N-cadherin expression on human Pca cell lines, Du-145 and PC-3 by flow cytometry. TNF-alpha, IL-10 and IL-12 were also evaluated. ICAM-1, expressed on PC-3 and DU 145 cells, was enhanced by TNF-alpha and IL-10. ELAM-1 was present on DU 145 cells but absent on PC-3. TNF-alpha and IL-10 enhanced ELAM-1 on DU 145, but EosA 24 hr supematants failed to do so. All three cytokines, namely IL-10, IL-12 and TNF-alpha-induced ELAM-1 on PC-3 tumor cells. Although VCAM-1 was absent on DU 145 and PC-3 cells, it was expressed on DU-145 cells after exposure to EosA: tumor cell co-cultures, and was expressed on PC-3 following exposure to IL-10 and IL-12. N-cadherin and E-cadherin were both expressed on DU-145. While N-cadherin was expressed on PC-3 cells, E-cadherin was not. N-cadherin was enhanced on DU-145 and PC-3 cells following exposure to EosA co-culture and upregulated on PC-3 by IL-10 and EosA 24 hr supernatants, but decreased by IL-12. E-cadherin was up-regulated on DU 145 cells following co-culture with EosA and was induced on PC-3 by IL-10 and IL-12, but not by EosA co-culture and 24 hr supematants. In conclusion, inflammatory and non-inflammatory cytokines modulate CAM expression on Pca cells; EosA and EosA 24 hr supernatants also exerted modulatory activity of CAM expression. Most significantly, the metastasis suppressor molecule, E-cadherin was enhanced on DU 145 cells by EosA and induced on PC-3 by IL-10 and IL-12 both of which are produced by EosA. This suggests potential use of these cytokines in immunotherapeutic strategies for prostate cancer and its metastasis.

Coadministration of swainsonine and doxorubicin attenuates doxorubicin-induced lethality in mice.

This study in mice concerns the protective effectiveness and mechanisms of action by which a coadministered regimen of an immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) protects against lethality induced by a single bolus intraperitoneal injection of LD50/14 doxorubicin. This swainsonine coadministration treatment regimen has been identified previously in our laboratory as the superior of the two optimal conditions for diminishing lethality in mice due to LD50/14 doxorubicin. The anthracycline, doxorubicin is a potent and widely used cancer chemotherapeutic agent whose clinical usefulness is limited by both a dose- and time-dependent cardiomyopathy. Specifically, mice were given simultaneous injections of swainsonine or its diluent buffer, phosphate buffered saline and LD50/14 doxorubicin on day 0, followed by twice daily injections of swainsonine or phosphate buffered saline up to day +9. The survival and well being of mice were monitored daily for 70 days, which may be considered equivalent to a period of 4 to 5 years in humans. This duration has a clinical implication with respect to the late manifestation of cardiotoxicity after doxorubicin treatment. We quantified the bone marrow cellularity of mice and performed in vitro progenitor cell assays to determine the effects of swainsonine coadministration treatment regimen on bone marrow competence after doxorubicin treatment. The effects of this regimen on doxorubicin-induced changes in heart morphology and on hematologic toxicities caused by doxorubicin were determined. This swainsonine coadministration treatment regimen significantly diminished doxorubicin-induced lethality and prolonged survival and well being of mice by preventing bone marrow pancytopenia from the start of therapy. It decreased bone marrow toxicity and facilitated its restoration. It accelerated restoration of blood hematocrit and total leukocyte levels. Also it facilitated the proliferation and differentiation of bone marrow pluripotent stem cells along the different paths to progenitor lineages, and significantly preserved the mouse heart morphology. These underlying mechanisms of action for the protection by swainsonine coadministration strongly suggest a potential role for swainsonine in high dose chemotherapy with doxorubicin.

Eosinophils in a tri-cell multicellular tumor spheroid (MTS)/endothelium complex.

Eosinophils have been found in infiltrates of many different cancers. It is still unclear as to whether they are passive bystanders in the cellular milieu or active cellular agents in host responses. Thus their harmful or helpful nature remains equivocal. We have developed an in vitro tri-cell model of eosinophils, MCF-7 breast tumor cell spheroids and HUVEC endothelial cells to examine the binding and association of eosinophils with both the tumor and the endothelia and the ensuing action of the tumor. Eosinophils bound very rapidly to the tumor spheroid and remained tightly bound throughout the 24 hr culture period. Histological staining of the tri-cell complex revealed highly granulated eosinophils as well as large amounts of degranulated protein diffused throughout the spheroid. IL-5 treatment of eosinophil: MTS complexes resulted in destruction of the tumor cells, particularly those which had grown out from the spheroid onto the endothelial cells. Eosinophils, pretreated with IL-5 before interaction with the tumor or endothelial cells, bound aggressively to the endothelial cells, thereby preventing tumor attachment. This eosinophil tri-cell tumor model system mimics clinical observations with regards to binding to epithelial and endothelial cells, dispersal of granular proteins throughout the tumor and also tumor destruction. Because it closely mirrors in vivo cellular interactions, it allows one to study more closely the mechanism(s) of eosinophil killing, the modulation of eosinophil activity and the testing of therapeutic interventions. The accommodation of the model to tumor invasion, using metastatic tumor cells and extracellular matrices such as matrigel, will help to elucidate a role for eosinophils (and their mediators) in cancer invasion and metastasis.

Mice primed with swainsonine are protected against doxorubicin-induced lethality.

The anthracycline, doxorubicin is a potent cancer chemotherapeutic agent whose therapeutic usefulness is limited by both a dose- and time-dependent cardiomyopathy. We tested the ability of an immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) to protect C57BL/6 mice against lethality within 70 days following a single bolus intraperitoneal injection of LD50/14 doxorubicin. Also, we sought the potential mechanisms responsible for this protection. This extended 70-day study in mice, which may be considered equivalent to a period of 4 to 5 years in humans, has clinical implication for delayed cardiotoxic sequela of therapy with high dose doxorubicin. Mice were pretreated with swainsonine or its diluent buffer, phosphate buffered saline for ten consecutive days prior to a single bolus intraperitoneal injection of a LD50/14 doxorubicin. We have previously defined this swainsonine pretreatment regimen as one of the two optimal conditions for swainsonine rescue of mice from death induced by LD50/14 doxorubicin. The survival and well being of groups of mice pretreated with swainsonine and phosphate buffered saline prior to LD50/14 doxorubicin, sham-treated and untreated were monitored daily for up to 70 days. The bone marrow cellularity of the mice were quantified, and in vitro progenitor cell assays were used to determine the effects of these treatment regimens on bone marrow competence following doxorubicin treatment. The effects of these treatment regimens on heart morphology and hematologic toxicities were also determined. This swainsonine pretreatment regimen significantly abrogated doxorubicin-induced lethality and prolonged survival of mice by facilitating restoration of bone marrow cellularity, accelerating restoration of blood hematocrit and total leukocyte levels, enhancing the proliferation and differentiation of bone marrow pluripotent stem cells along the different paths to progenitor lineages, and preserving the heart morphology. This study strongly suggests a potential role for swainsonine with doxorubicin in cancer chemotherapy.

Recruitment experience in the first phase of the African American Hereditary Prostate Cancer (AAHPC) study.

The African American Hereditary Prostate Cancer (AAHPC) Study is an ongoing multicenter genetic linkage study organized by Howard University and the National Human Genome Research Institute (NHGRI), with support from the Office for Research on Minority Health and the National Cancer Institute. The goals of the study are to: (i) look for evidence of involvement of chromosome 1q24-25 (HPC1) in African American men with hereditary prostate cancer (HPC) and (ii) conduct a genome-wide search for other loci associated with HPC in African American men. To accomplish these goals, a network has been established including Howard University, the NHGRI, and six Collaborative Recruitment Centers (CRCs). The CRCs are responsible for the identification and enrollment of 100 African American families. To date, 43 families have been enrolled. Recruitment strategies have included mass media campaigns, physician referrals, community health-fairs/prostate cancer screenings, support groups, tumor registries, as well as visits to churches, barber shops, and universities. By far, the most productive recruitment mechanisms have been physician referrals and tumor registries, yielding a total of 35 (81%) families. Approximately 41% (n = 3400) of probands initially contacted by phone or mail expressed interest in participating; the families of 2% of these met the eligibility criteria, and 75% of those families have been enrolled in the study, indicating a 0.5% recruitment yield (ratio of participants to contacts). As the first large-scale genetic linkage study of African Americans, on a common disease, the challenges and successes of the recruitment process for the AAHPC Study should serve to inform future efforts to involve this population in similar studies.

Loss of leukoregulin up-regulation of natural killer but not lymphokine-activated killer lymphocytotoxicity in human papillomavirus 16 DNA-immortalized cervical epithelial cells.

The sensitivity of human cervical epithelial cells immortalized by transfection with human papillomavirus type 16 (HPV16) DNA, to lysis by natural killer (NK) and lymphokine-activated killer (LAK) lymphocytes was evaluated at progressive stages of transformation. Both early- (10-20 wk) and late- (greater than 30 wk) passage HPV16-immortalized cells were resistant to NK lymphocyte cytotoxicity but sensitive to LAK lymphocyte cytotoxicity at lymphocyte-to-cervical cell ratios ranging from 1:1 to 50:1 in a 4-hour 51Cr release assay. Treatment of early-passage HPV16 DNA-immortalized cells with 2.5 U/mL of the NK lymphocytotoxicity-sensitizing lymphokine, leukoregulin, for 1 hour induced modest sensitivity to NK cells (P less than .05) but markedly up-regulated LAK sensitivity twofold to threefold. At the later passages, leukoregulin up-regulation of sensitivity to NK was lost but remained to LAK lymphocytotoxicity. Similarly, an HPV16-positive human cervical carcinoma cell line, QGU, was also resistant to NK lymphocytotoxicity and sensitive to LAK lymphocytotoxicity; leukoregulin failed to confer sensitivity to the NK-resistant QGU tumor cells and increased their sensitivity to LAK lymphocytotoxicity 1.5-fold to twofold. Although the HPV-immortalized cervical cells containing integrated HPV16 DNA were not tumorigenic, they mimicked the response of established HPV16-positive cervical carcinoma cells. HPV16-immortalized cervical epithelial cells provide a useful model for the study of cytokine modulation of dysplastic and neoplastic cervical epithelial cell sensitivity to natural lymphocytotoxicity.

Leukoregulin up-regulation of tumor cell sensitivity to natural killer and lymphokine-activated killer cell cytotoxicity.

The present investigation demonstrates that leukoregulin, a cytokine secreted by natural killer (NK) lymphocytes up-regulates the sensitivity of tumor cells to lymphokine-activated killer (LAK) cell cytotoxicity. It has been previously established that leukoregulin increases the sensitivity of sarcoma, carcinoma and leukemia cells to natural killer (NK) cell cytotoxicity. Tumor cells were treated with leukoregulin for 1 h at 37 degrees C and tested for sensitivity to NK and LAK cytotoxicity in a 4-h chromium-release assay. NK-resistant Daudi, QGU and C4-1 human cervical carcinoma cells became sensitive to NK cytotoxicity after leukoregulin treatment, and their sensitivity to LAK was increased two- to sixfold. Y-79 retinoblastoma cells, which are moderately sensitive to NK and very sensitive to LAK, became increasingly sensitive (two- to four-fold) to both NK and LAK cell cytotoxicity. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant interleukin-1 (alpha and beta), recombinant interferon gamma, recombinant tumor necrosis factor or combinations of the latter two failed to up-regulate tumor cell sensitivity to NK and LAK cell cytotoxicity. However, treatment with recombinant interferon gamma for 16-18 h, GM-CSF and interleukin-1 beta for 1 h induced a state of target cell resistance to both NK and LAK cell cytotoxicity. Leukoregulin may have an important physiological function in modulating NK and LAK cell cytotoxicity by increasing the sensitivity of target cells to these natural cellular immunocytotoxicity mechanisms.