Marek Mirowski

Functional C3435T polymorphism of MDR1 gene: an impact on genetic susceptibility and clinical outcome of childhood acute lymphoblastic leukemia.

The significance of genetic background in childhood acute lymphoblastic leukemia (ALL) is not well understood. Polymorphisms of genes encoding for xenobiotics and drug transporters are potential factors, which can influence the risk of developing ALL and its clinical outcome. P‐glycoprotein (P‐gp) is an adenosine triphosphate‐binding cassette (ABC)‐family transporter involved in protection against xenobiotics and multi‐drug resistance. Recently, the single‐nucleotide polymorphism C3435T of MDR1 gene has been found to be associated with altered tissue expression and function of P‐gp. To evaluate whether C3435T MDR1 polymorphism is associated with the occurrence and outcome of ALL, 113 children with ALL (median age 5.1 yr) and 175 healthy individuals of Polish Caucasian origin were studied by polymerase chain reaction‐restriction fragment‐length polymorhism (PCR‐RFLP) assay. The mutant homozygous TT genotype was found to be associated with occurrence of ALL (OR, 95% CI; 1.8, 1.1–3.1; P = 0.037). Besides, the analysis of factors influencing clinical outcome of our ALL patient cohort showed that CC genotype carriers had significantly lower event‐free survival probability (pEFS) (0.62 vs. 0.87; P = 0.007) and overall survival probability (pOS) (0.72 vs. 0.91; P = 0.006). The Cox proportional hazards model‐based analysis revealed that the hazard ratios for lower pEFS and lower pOS among CC homozygous subjects were 3.9 (P = 0.008) and 3.3 (P = 0.02), respectively. In conclusion, the results of the present study provide evidence that C3435T MDR1 polymorphism may involve both the susceptibility to and the clinical outcome of childhood ALL. Carriers of the TT genotype are more at risk of developing ALL than other individuals, whereas CC genotype carriers are supposed to have worse prognosis.

ABCB1/MDR1 gene polymorphisms as a prognostic factor in colorectal cancer

ObjectiveTo analyse the single-nucleotide polymorphisms (SNPs): ABCB11236C>T, ABCB12677G>T/A, ABCB13435C>T and haplotypes in the ABCB1/MDR1 gene, which could contribute to genetic risk of colorectal cancer (CRC). Disease association between the ABCB1/MDR1 genotype, allele, haplotype frequencies and histological features, such as TNM classification, localization of primary carcinoma, grade of malignancy, histological type of tumour, lymphoid infiltration and vessel invasion were estimated. In this study, the potential role of SNPs of the ABCB1/MDR1 gene as a prognostic marker for CRC was analysed.Materials and methodsTumour specimens of 95 patients with CRC were studied. Using automated sequencing or PCR-RFLP method, DNA for three common SNPs of ABCB1/MDR1 was extracted and analysed. The results of genotyping and haplotype analysis with histopathological features, grading and clinical staging of neoplasms were correlated.ResultsA statistically significant higher frequency of T1236 allele in T1/T2 (89.7%), M0 groups (81.6%) and I/II clinical staging (82.7%) in comparison with T3/T4 (68.2%), M1 groups (47.4%) and III/IV clinical staging (65.1%) was detected. Furthermore, multivariate analysis according to Coxs proportional hazard model indicated that the T1236 allele is a good, independent prognostic factor and the presence of this allele decreases the risk of death in comparison with a group without this allele (HR = 0.26; p = 0.0424). In addition, a statistically significant higher frequency of C3435 allele and significant differences in the C3435 allele distribution in N1/N2 group (91.7% and 62.5%, respectively) than N0 group (71.2% and 44.9%, respectively) was found. Each of the eight possible haplotypes was noted in M0 or I/II group and only seven in M1 or III/IV group. Haplotype T1236-G2677-C3435 only in less advanced CRC subjects (9.6% in I/II and 9.2% in M0 group) was detected. In addition, significant differences in haplotype distributions between M0 or I/II and M1 or III/IV group were found (p = 0.01 and p = 0.05, respectively).ConclusionsThese results suggest association between T1236 allele and T1236-G2677-C3435 haplotype and less advanced CRC, so these genetic markers may play a role as potentially good prognostic factors. Differences in haplotype distributions and degree of clinical staging may suggest that some other potential SNPs, especially in regulatory region of ABCB1/MDR1 gene, may influence P-glycoprotein function and CRC progression.

Polymorphisms and haplotypes in the multidrug resistance 1 gene (MDR1/ABCB1) and risk of multiple myeloma

MDR1(ABCB1) gene encodes for P-glycoprotein (P-gp, MDR1, ABCB1), an ATP-binding cassette superfamily member involved in the transport of xenobiotics. Here, we investigated whether common MDR1 single nucleotide polymorphisms (1236C>T, 2677G>A/T and 3435C>T) affect predisposition to multiple myeloma. Genotyping was performed in 111 myeloma patients and 96 controls by PCR-based assays. Haplotypes were inferred using PHASE algorithm. We found comparable allele and genotype frequencies among myeloma patients and controls. Moreover, patient and control groups did not differ regarding MDR1 haplotype distribution (p=0.18). In conclusion, our results do not support major influence of MDR1 variants on the risk of myeloma in Caucasians.

CD38 Gene Polymorphisms Contribute to Genetic Susceptibility to B-Cell Chronic Lymphocytic Leukemia: Evidence from Two Case-Control Studies in Polish Caucasians

Given the recent findings on the importance of CD38 signaling in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL), we hypothesized that single nucleotide polymorphisms (SNP) in the CD38 gene may be related to B-CLL risk. We evaluated two potentially functional CD38 SNPs, intronic rs6449182 (184C>G) and missense rs1800561 (418C>T, Arg140Trp) in two hospital-based case-control studies (study A and validation study B). Genotyping was done using PCR-based assays in a total of 460 Polish Caucasian patients with B-CLL and 503 age-matched and gender-matched controls. We found that frequencies of both variant alleles (rs6449182 G and rs1800561 T) were significantly higher in B-CLL. In study A, logistic regression analysis revealed an association between B-CLL and genotypes: rs6449182 CG [odds ratio (OR), 3.57; 95% confidence interval (95% CI), 2.4-5.3], rs6449182 GG (OR, 5.2; 95% CI, 2.36-11.5), and rs1800561 CT (OR, 6.72; 95% CI, 1.5-30.1), although no homozygous rs1800561 TT genotype was detected in either study. These results were confirmed in study B, which showed an association between B-CLL and genotypes rs6449182 CG (OR, 4.00; 95% CI, 2.7-6.0), rs6449182 GG (OR, 12.84; 95% CI, 4.3-38.7), and rs1800561 CT (OR, 10.12; 95% CI, 1.3-81.6), and in the combined analysis of both studies. We also observed that rs6449182 G carriers had more advanced clinical stage (P = 0.002) and tended to be younger at diagnosis (P = 0.056). Furthermore, we found higher CD38 transcript levels and higher proportions of CD38-positive cells in carriers of rs6449182 G and rs1800561 T alleles (P < 0.05 for all comparisons). In conclusion, our data show that CD38 SNPs may affect CD38 expression and contribute to the increased risk of B-CLL carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2009;18(3):945–53)

No influence of 3435C>T ABCB1 (MDR1) gene polymorphism on risk of adult acute myeloid leukemia and P-glycoprotein expression in blast cells

Inherited differences in xenobiotic transport and metabolism may play an important role in the development of adult acute myeloid leukemia (AML) and response to the chemotherapy. An ATP-binding cassette (ABC) family transporter P-glycoprotein (P-gp or ABCB1), encoded by ABCB1 (MDR1) gene, is involved in the protection against xenobiotics and multi-drug resistance. The aim of this study was to investigate the potential involvement of the ABCB1 gene exon 26 3435C>T single nucleotide polymorphism (SNP) in the genetic susceptibility to AML and regulation of P-gp expression and activity in AML cells. A total of 180 adult AML patients and 180 sex-matched controls were genotyped using PCR-RFLP method. Moreover, in 40 AML patients ABCB1 gene expression was studied by real-time RT-PCR and P-gp expression and activity were assessed by flow cytometry assays. The prevalence of 3435C>T ABCB1 polymorphism was similar in patient and control cohorts (P = 0.16). Furthermore, the carriers of different ABCB1 genotypes did not differ significantly according to ABCB1 gene expression (P = 0.99), P-gp expression (P = 0.42) and P-gp activity (P = 0.83) in leukemic cells. The authors conclude that isolated 3435C>T ABCB1 SNP is not a major factor of the genetic susceptibility to adult AML, and that genotyping of this polymorphism does not allow predicting P-gp expression or activity in AML cells.

HMGI(Y) gene expression in colorectal cancer: comparison with some histological typing, grading, and clinical staging.

We investigated HMGI(Y) gene expression in 81 pairs of frozen samples obtained from colorectal carcinomas and adjacent normal colorectal mucosas and in four samples from colorectal mucosa from patients without neoplastic diseases. In this group, HMGI(Y)-positive/-negative expression was compared with some histological features, grading, and clinical staging of neoplasms investigated to assess its potential role as a prognostic marker for colorectal cancer. Expression of HMGI(Y) gene was found in 51 of 81 cases of colorectal cancers, while, in normal mucosa, expression of this gene was not observed. HMGI(Y) gene expression was associated with more advanced tumors (T3, T4) and metastases to lymph nodes (N1, N2). The most interesting finding was that expression of this gene correlated with distant metastases. HMGI(Y) gene expression was detected in all cases classified as M1 (n = 19, p = 0.0008). We did not find any association between age, gender, tumor localization, histological type and this gene expression.

Structure-activity Relationship, Conformation and Pharmacology Studies of Morphiceptin Analogues - Selective μ-Opioid Receptor Ligands

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) is one of the most selective agonists for the mu-opioid receptor. In this review structure-activity relationships of morphiceptin analogues and studies resulting in defining low energy conformations are discussed. Finally, new developments in the control of tumour growth and cell proliferation by morphiceptin analogues are surveyed, which open future perspectives in the diagnosis and treatment of various cancers.

C3435T polymorphism of the ABCB1 gene: impact on genetic susceptibility to peptic ulcers

The functional single nucleotide polymorphism (SNP) C3435T in exon 26 of the ABCB1 gene encoding the xenobiotic transporter P-glycoprotein (P-gp) may influence susceptibility to several diseases, as well as the clinical outcome of treatment with P-gp substrates. Exposure to environmental chemicals is thought to be involved in peptic ulcer pathogenesis and then later in stomach cancer development. About 80% of ulcers are associated with Helicobacter pylori infection, one of the risk factors of stomach cancer. P-gp-transported drugs are used in treatment of H. pylori. Therefore, a lack of effectiveness in eradication therapy can lead to chronic stomach inflammation and promote cancerogenesis. In this study, 196 patients with peptic ulcers divided into two groups with and without H. pylori infection and combined with 96 healthy controls were genotyped for the ABCB1 C3435T SNP. A trend towards higher incidence of the 3435TT genotype among peptic ulcer patients than in controls (p = 0.0983) was observed. Likewise, the 3435T allele was more frequent in groups suffering from peptic ulcers. The association was near to statistical significance (p = 0.0538). Between analyzed genotypes and H. pylori infection, statistically significant dependence was found (p = 0.0372). In addition, the CT genotype was associated with 1.56 times and the TT with 2.45 times higher prevalence of infection compared to the CC genotype. Asimilar association was present in a subgroup of peptic ulcer men (p = 0.0090). The isolated C3435T ABCB1 SNP is not a major factor for genetic susceptibility to peptic ulcer, but in a group of men who suffered from peptic ulcer, this polymorphism seemed to be a risk factor for H. pylori infection development.

Characterization of the [125I]endomorphin-2 binding sites in the MCF7 breast cancer cell line.

In the present study, the expression of the micro-opioid receptor on protein level has been demonstrated in MCF7 breast cancer cells. Binding of the [125I]-labeled micro-opioid receptor selective ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) was examined in vitro using a cross-linking assay followed by a Western blot technique. The radioactive complex had a molecular weight of about 65 kDa and was detectable by anti-micro-opioid receptor antibody, indicating the presence of micro-opioid receptors in MCF7 cell membranes. Characterization of endomorphin-2 binding to the membranes obtained from MCF7 cells was performed. Cold saturation experiments with [125I]endomorphin-2 showed biphasic binding curves in Scatchard coordinates. One component represents a high affinity and low capacity, and the other low affinity and higher capacity binding sites. The obtained Bmax values for [125I]endomorphin-2 binding to MCF7 membranes were much higher than those obtained for mouse brain. Pharmacological characterization of the [125I]endomorphin-2 binding sites was made using endomorphin-2 and two other micro selective ligands, morphiceptin, and [D-1-Nal3]morphiceptin on MCF7 cell membrane preparations and whole MCF7 cells. In both cases, the rank order of potency was [D-1-Nal3]morphiceptin>endomorphin-2>morphiceptin, but in case of whole MCF7 cells the IC50 values were about 40 times higher.

Binding of the new morphiceptin analogs to human MCF-7 breast cancer cells and their effect on growth

In the present study, we reported on the synthesis of two new mu-opioid peptide analogs, [D-1-Nal3]morphiceptin and [D-1-Nal4]-morphiceptin [1-Nal=3-(1-naphthyl)-alanine] which expressed receptor binding affinities at least at the level of the primary opioid ligands. The new analogs also labeled mu-opioid receptors on the cells of human breast cancer MCF-7 cell line with affinity much higher than that of endomorphins and morphiceptin, the well-known mu-selective opioid peptides. However, none of the tested peptides significantly decreased cell proliferation of MCF-7 cells.