Andrzej Roszak

Folate and choline metabolism gene variants and development of uterine cervical carcinoma

OBJECTIVE It has been reported that aberrant DNA methylation can be associated with HPV infection and cervical tumorigenesis. The aim of this study was to evaluate the possibility that polymorphic variants of genes that may affect DNA methylation status are associated with the risk of cervical cancer in the Polish population. DESIGN AND METHOD Employing PCR-RFLPs and HRM analyses, we examined the prevalence of BHMT Arg239Gln (rs3733890), MTR Asp919Gly (rs1805087), MTHFR Ala222Val (rs1801133), MTHFD1 Arg653Gln (rs2236225) and MTRR Ile22Met (rs1801394) genotypes and alleles in patients with advanced cervical cancer (n=124) and controls (n=168). RESULTS The odds ratio (OR) for BHMT Gln/Gln genotype was 0.433 (95% CI=0.1780-1.054; p=0.0602). The OR for patients having the BHMT Arg/Gln or Gln/Gln genotypes was 0.579 (95% CI=0.3622-0.924; p=0.0216). We also observed a significantly higher frequency of the BHMT 239Gln allele in controls than in patients, p=0.0165. The genotype and allele frequencies of the MTR Asp919Gly, MTHFR Ala222Val, MTHFD1 Arg653Gln and MTRR Ile22Met gene variants did not display statistical differences between patients with cervical cancer and controls. We also did not find a significant association between the distribution of any genotypes or alleles and cancer characteristics. CONCLUSION Our results might suggest the protective role of the BHMT 239Gln variant in cervical cancer incidence.

Monocyte Chemoattractant Protein-1−2518 A/G Single Nucleotide Polymorphism Might Be Associated with Renal Disease and Thrombocytopenia of SLE

There is conflicting evidence on the contribution of the MCP-1 −2518 A>G (rs 1024611) polymorphism to SLE incidence and clinical manifestations. We examined the prevalence of the MCP-1 −2518 A>G polymorphism in SLE patients (n = 199) and controls (n = 250) in Poland. We did not observe a significant difference in the distribution of MCP-1 −2518 A>G polymorphic variants in patients with SLE and healthy individuals. However, we found an association between the GG versus AG and AA genotypes as well as the AG and GG versus AA genotypes with renal manifestations of SLE OR = 3.614 (1.123–11.631, P = 0.0345) and OR = 2.297 (1.301–4.057, P = 0.0046), respectively. We also observed that the MCP-1 AG and GG -genotypes contribute to the occurrence of thrombocytopenia in SLE patients OR = 2.618 (1.280–5.352, P = 0.0089). Our observations indicate that either MCP-1 −2518 G variant can be associated with some clinical findings in patients with SLE.

Involvement of the XRCC1 Arg399Gln gene polymorphism in the development of cervical carcinoma

Background Although infection with the human papillomavirus (HPV) is crucial to the development of cervical cancer, it is not considered a sufficient isolated factor to cause this malignancy. The association of the XRCC1 Arg399Gln (rs25487) polymorphism with cervical cancer has been demonstrated in some populations. Methods The XRCC1 Arg399Gln genetic variants were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in patients with advanced cervical cancer (n=189) and controls (n=308). Results We observed that patients with advanced cervical cancer having the Gln/Gln or Gln/Arg vs Arg/Arg genotype displayed a 1.726-fold increased risk of cervical cancer (95% confidence interval [CI]=1.158-2.572, p=0.007). The odds ratio (OR) for Gln/Gln vs Gln/Arg or Arg/Arg was 1.742 (95% CI=1.073-2.827; p=0.0236). We also found a significantly higher frequency of the XRCC1 399Gln allele in patients with cancer than in controls, with OR=1.489 (95% CI=1.148-1.930, p=0.0026). The p value of the chi-square test for the trend observed for the XRCC1 Arg399Gln polymorphism was also statistically significant (ptrend=0.002). The statistical power of this study amounted to 78% for the Gln/Gln or Gln/Arg genotypes and 61% for the Gln/Gln genotype. Conclusion Although the statistical power of our study did not reach 80%, we found a statistically significant association between the XRCC1 399Gln variant and the incidence of cervical cancer.

Transcriptional analysis of CXCR4, DNMT3A, DNMT3B and DNMT1 gene expression in primary advanced uterine cervical carcinoma.

The development of cervical cancer requires genetic and epigenetic factors which result in the persistence of a malignant phenotype. Cervical cancer exhibits also some unique differences from other solid tumors. Normal cervical stratified epithelia have characteristics of hypoxic tissue with over-expression of HIF-1 (hypoxia-inducible factor-1) transcription factor, which targets the transcription of over 70 genes involved in many aspects of cancer biology. One of the genes, which could be induced by HIF-1 is chemokine (C-X-C motif) receptor 4 (CXCR4). CXCR4 could also be epigenetically regulated by methylation of CpG dinucleotides located in the promoter region. Here, we examined the CXCR4, DNMT3A, DNMT3B and DNMT1 transcript levels in cancer tissue (n=30) and non-cancer, normal uterine cervical tissue (n=30) from a Polish cohort. We also compared the methylation status of CXCR4 promoter region in cancer and normal tissue samples. Our result showed significantly higher levels of CXCR4, DNMT3A, DNMT3B and DNMT1 transcript (p=0.0058, 0.0163, 0.0003 and <0.0001, respectively) levels in cancer tissue as compared to normal samples. We did not observe DNA methylation in the CXCR4 promoter region in either control or cancer tissue samples. CXCR4 has a functional hypoxia response element (HRE) in the promoter region, located -1.3 kb from the transcription start site. Our work shows for the first time that HIF-1A could promote the induction of CXCR4 gene expression (Spearmans correlation coefficient = 0.515, p=0.003) in patients with primary advanced uterine cervical carcinoma.

HER2 codon 655 polymorphism is associated with advanced uterine cervical carcinoma.

OBJECTIVES It has been suggested that overexpression of HER2 in advanced cervical tumors can be considered an independent predictor of poor patient outcome. DESIGN AND METHODS Employing PCR-RFLPs, we examined the distribution of HER2 Ile655Val (rs 1136201) genotypes and alleles in patients with advanced cervical cancer (n=109) and controls (n=220). RESULTS Odds ratio (OR) for patients with advanced cervical cancer with the HER2 Val/Val homozygous or Val/Ile heterozygous state was 1.778 (95% CI=1.117-2.830, p=0.0176). We also observed an association of the HER2 Val/Val genotype with advanced cervical cancer in the patient group OR=3.706 (95% CI=1.061-12.950, p=0.0459). However, we did not find a significant association between the distribution of genotypes or alleles and cancer characteristics for the HER2 Ile655Val polymorphism. CONCLUSIONS Our results indicate that the HER2 655Val variant may be associated with the incidence of advanced cervical cancer.

Increased expression of HIF-1A and its implication in the hypoxia pathway in primary advanced uterine cervical carcinoma.

The development of cervical cancer exhibits some unique differences compared to other solid tumors. Normal cervical stratified epithelia have characteristics of hypoxic tissue. Lack of oxygen (hypoxia) induces the HIF-1 (hypoxia-inducible factor-1) transcription factor, which is a heterodimer composed of a constitutively expressed β subunit and a hypoxia-inducible α-subunit. HIF-1A targets the transcription of over 70 genes involved in many aspects of cancer biology. In well-oxygenated environments, the HIF-1A subunit is hydroxylated, recognized and marked for proteosomal destruction by an E3 ubiquitin ligase, the von Hippel-Lindau protein (pVHL) complex. Under hypoxic stress, proline hydroxylase (PHD) activity is diminished, and stabilized HIF-1A is involved in the activation of the tissue response to hypoxia. Here, we examined the HIF-1A and VHL transcript levels and HIF-1A protein levels in cancerous tissue (n=30) and non-cancerous, normal uterine cervical tissue (n=30). We also compared the methylation status of HIF-1A and of the VHL promoter regions in cancerous and normal tissue samples. Significantly higher levels of HIF-1A and VHL transcripts (p<0.0001 and p=0.0042, respectively) and of HIF-1A protein (p=0.0037) were detected in cancerous tissue compared to normal samples. We did not observe DNA methylation in the HIF-1A and VHL promoter region in either control or cancerous tissue samples. VHL has a functional hypoxia response element (HRE) in the promoter region, and the induction of this HRE acts within a negative feedback loop to limit the hypoxic HIF-1A response. Our findings may suggest that HIF-1A could promote its own degradation by the induction of VHL gene expression (Spearman correlation coefficient, 0.515; p=0.003). Our study shows for the first time that this increase in VHL expression could be HIF-1A-dependent and serves within a negative feedback pathway during hypoxia to regulate the cell-specific oxygen threshold for HIF-1A activation.

Distribution of CCND1 A870G Polymorphism in Patients with Advanced Uterine Cervical Carcinoma

We examined the distribution of the CCND1 A870G (rs9344) polymorphic variant in patients with cervical cancer (n = 129) and healthy individuals (n = 288) in a sample of a Polish cohort. We showed that patients with advanced cervical cancer bearing the CCND1 A/A and A/G genotypes displayed a 1.811-fold increased risk of cervical cancer (95% CI = 1.150–2.852, p = 0.0098). We also found a significantly higher frequency of the CCND1 870A allele in patients with cancer than in controls, p = 0.0116. Our investigation confirmed that the CCND1 870A gene variant may be a genetic risk factor in the incidence of advanced cervical cancer.

Prevalence of the NKG2D Thr72Ala Polymorphism in Patients with Cervical Carcinoma

BACKGROUND The natural killer group 2, member D (NKG2D) receptor is mainly situated on the surface of NK and CD8(+) αβ T cells that are involved in the defense against viral agents and in cancer immunosurveillance. The G>A transition (Thr72Ala) (rs2255336) located in the NKG2D region encoding the transmembrane part of this receptor has been associated with decreased functionality of NK and T cells. METHODS Using polymerase chain reaction-restriction fragment length polymorphisms, we examined the NKG2D Thr72Ala polymorphism in patients with cervical cancer (n=353) and controls (n=366) in a Polish population. RESULTS We observed an increased frequency of Thr/Thr or/and Thr/Ala genotypes in controls compared with all patients with cervical cancer; however, these differences were not significant. We found a significantly increased frequency of the NKG2D 72Thr allele in controls than in all patients (odds ratio [OR]=0.7410 [95% confidence intervals (CI)=0.5683-0.9662, p=0.0265]). Moreover, stratification of patients based on cancer stage showed a significant increase in the Thr/Thr genotype frequency (OR=0.3086 [95% CI=0.09097-1.047, p=0.0461]), as well as in the Thr/Thr and Thr/Ala genotype frequency (OR=0.4504 [95% CI=0.2891-0.7018, p=0.0003]), in controls compared with patients with cervical cancer in stages III and IV. The frequency of the NKG2D 72Thr allele was also significantly increased in controls as compared with patients in stage III and IV cancer (OR=0.4699 [95% CI=0.3170-0.6967, p=0.0001]). CONCLUSION Our studies may suggest that the women with cervical cancer bearing the NKG2D 72Thr gene variant might be protected against progression to advanced stages of this cancer.

Urinary tract infections in patients with malignant neoplasms of the genitourinary system

Summary Aim To diagnose the etiological factors involved in urinary tract infections (UTI) and to examine antibiotic sensitivities in bacteria – causative agents of UTI in female patients with genitourinary malignancies. Materials/Methods Nineteen patients aged between 35 and 75 years treated for genitourinary malignancies and with a diagnosis of UTI were examined. The majority of these patients (17) were treated with X rays to the pelvic region (tele – and brachytherapy). Six patients were treated for endometrial carcinoma. One patient was treated for ovarian cancer, and one for vaginal cancer. Significant bacteriuria was defined as 10 4 or more CFUs/ml in quantitative urine examination. Microorganisms were identified according to standard bacteriological methods using bio-Merieux tests. The isolated bacteria were tested for antibiotic sensitivity using the ATB Expression system. Results E. coli was the most frequently observed causative factor in cases of UTI. E. faecalis strains were the next most commonly observed species. One of 11 isolated E. coli strains was resistant to 5 antibiotics, a second was resistant to 3 antibiotics and three strains were resistant to 2 antibiotics. E. coli strains were classified into 9, and E. faecalis into 4, sensitivity patterns. UTI was diagnosed in four patients with stage I B, in four with stage III B and in one patient with stage IV cervical cancer. Conclusions E. coli and E. faecalis were the most frequently isolated species from the urine of female patients with genitourinary malignancies. These bacteria are among the main causative agents of UTI. The results of these investigations may be used for epidemiological purposes: to find the causes and sources of nosocomial infections and in the prophylaxis of these infections.

Increased 17ß-hydroxysteroid dehydrogenase type 1 levels in primary cervical cancer

Infections with oncogenic human papillomavirus (HPV) strains are recognized as the major risk factor for developing malignant lesions in the uterine cervix. However, several findings have demonstrated cooperation between HPV infection and 17β-estradiol (E2) in cervical carcinogenesis. The 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) is the enzyme involved in the transformation of estrone (E1) into E2. In this study, we identified the HSD17B1 transcript and protein in HeLa, SiHa, Ca Ski and C-33A cervical cancer cells. These cells were able to convert E1 to E2 in a time-dependent manner. Moreover, we identified the HSD17B1 transcript and protein in primary cancerous tissues (n=28) and in histologically unchanged tissues (n=25). We did not observe significant differences (P=0.33) between the HSD17B1 transcript levels in cancerous tissues and histologically unchanged tissues. However, we found an overrepresentation of the HSD17B1 protein in cancerous tissues compared with histologically unchanged tissues (P<0.001). This overrepresentation of the HSD17B1 protein in primary cervical cancerous tissues may be responsible for the local conversion of E1 to E2.