Marcela Mrhalova

Kinetics of heme interaction with heme-binding proteins: The effect of heme aggregation state

The kinetics of the interaction of heme with hemopexin and albumin was monitored by measuring the time dependence of changes in the Soret absorption spectra. Since the protein binding sites can only bind heme monomers, the binding kinetics apparently reflected the slow dissociation of heme dimers, resulting from dimer/monomer equilibria in aqueous heme solutions. The dissociation of heme dimers is characterized by the rate constant of (3-4) x 10(-3) s(-1). The measurements further revealed significant differences in the kinetic profiles (slowing down the binding interaction) that were dependent on the storage time of heme solutions at room temperature. These presumably responded to the gradual formation of higher aggregates of heme, which cannot dissociate into dimers/monomers. Alternatively, partial autooxidation of heme molecules could increase the stability of heme dimers and obstruct specific binding of heme to the proteins.

Breast cancer: role of polymorphisms in biotransformation enzymes.

We aimed at determining whether any association exists between genetic polymorphisms in epoxide hydrolase (EPHX1), NADPH-quinone oxidoreductase (NQO1), glutathione S-transferases (GSTM1/P1/T1) and individual susceptibility to breast cancer. Polymerase chain reaction-restriction fragment length polymorphism-based genotyping assays were used to determine the frequency of polymorphisms in EPHX1 (exons 3 and 4), NQO1 (exon 6), GSTM1 (deletion), GSTP1 (exon 5), and GSTT1 (deletion) in a case–control study comprised of 238 patients with breast cancer and 313 healthy individuals. The distribution of genotypes in exon 6 of NQO1 was significantly different between the control group and breast cancer cases. Age-adjusted odds ratio (OR) for variant genotype NQO1*2/*2 was 3.68 (confidence interval (CI)=1.41–9.62, P=0.008). Association of GSTP1*2/*2 genotype as well as that of low EPHX1 activity deduced by combinations of genotypes in exons 3 and 4 with breast cancer was suggestive, but nonsignificant. Individuals simultaneously lacking GSTM1 and carrying at least one GSTP1 variant allele were at significantly higher risk of breast cancer (OR=2.03, CI=1.18–3.50, P=0.010). Combinations of either GSTM1null or GSTP1*2 with low activity of EPHX1 presented significant risk of breast cancer (OR=1.88, CI=1.00–3.52, P=0.049 and OR=2.40, CI=1.15–5.00, P=0.019, respectively) as well. In conclusion, the results suggest that genetic polymorphisms in biotransformation enzymes may play a significant role in the development of breast cancer.

Association of superoxide dismutases and NAD(P)H quinone oxidoreductases with prognosis of patients with breast carcinomas

Associations of transcript levels of oxidative stress‐modifying genes SOD2, SOD3, NQO1 and NQO2 and their functional single nucleotide polymorphisms (SNPs) rs4880, rs1799895, rs2536512, rs699473, rs1800566 and rs1143684 with prognosis of breast cancer patients were studied. SNPs were assessed by allelic discrimination in a cohort of 321 breast cancer patients from the Czech Republic. Transcript levels were determined by real‐time polymerase chain reaction (PCR) with absolute quantification in tumor and adjacent non‐neoplastic control tissues. Both genotypes and transcript levels were then compared with available clinical data on patients. Patients carrying low activity allele Leu in NQO2 rs1143684 had a greater incidence of stage 0 or I disease (i.e., better prognosis) than patients with the Phe/Phe genotype. This association was more evident in patients without expression of progesterone receptors (p = 0.031). Patients carrying the Thr allele in SOD3 rs2536512 SNP had a significantly greater incidence of tumors expressing estrogen receptors than patients carrying the Ala/Ala genotype (p = 0.007). SOD3 transcript level was significantly higher in grade 1 or 2 tumors than in grade 3 tumors (p = 0.006). Patients carrying T allele in SOD3 rs699473 SNP had significantly poorer progression‐free survival (PFS) than patients carrying the CC genotype (p = 0.038). The same applied to the subgroup of patients treated by hormonal regimens (p = 0.021). Patients carrying the high activity Ala/Ala genotype in SOD2 (rs4880) had significantly poorer PFS than Val allele carriers in the group treated by cyclophosphamide but not hormonal regimens (p = 0.004). Our results suggest that NQO2, SOD2 and SOD3 may significantly modify prognosis of breast cancer patients and that their significance should be further characterized.

A novel quantitative PCR of proliferation markers (Ki-67, topoisomerase IIα, and TPX2): an immunohistochemical correlation, testing, and optimizing for mantle cell lymphoma

A clinical course of patients with mantle cell lymphoma (MCL) is aggressive, and the disease is rarely curable. Proliferation rate is the most important prognostic factor. We developed a novel, reliable, rapid, and routinely applicable approach allowing a precise quantitative assessment of three proliferation markers, Ki-67, topoisomerase IIα, and TPX2. A total of 95 lymphoma specimens were measured in the study by real-time reverse transcription PCR (RQ-RT-PCR). We tested the reproducibility and accuracy of the assay and correlated the results with the immunohistochemical staining of the corresponding proteins. The results obtained indicated individual variability of the mRNA expression levels, reflecting heterogeneity of the proliferation rate in individual patients. In general, we observed the highest mRNA expression in the group of Burkitt lymphomas and the lowest in patients with reactive lymphadenopathies. We found increased proliferation rate in MCLs with high cyclin D1 mRNA, indicating a quantitative control of the cell cycle. We observed a correlation between mRNA expression level and the immunohistochemical staining of corresponding proteins, which significantly argues for the prognostic significance of the mRNA expression measuring. We confirmed the accuracy of the current assay for a precise quantitative examination of the proliferation activity. Real-time RT-PCR provides a novel approach applicable for clinical trials, and it represents a potent approach allowing to stratify MCL patients for entry into clinical trials according to the expression of the proliferation signature genes in their tumors. This approach may contribute to improved and individualized therapeutic options respecting the individual progression risk of patients with MCL.

Single nucleotide polymorphisms in the multidrug resistance gene 1 (ABCB1): effects on its expression and clinicopathological characteristics in breast cancer patients.

Objectives Resistance of tumor cells to multiple cytostatic agents is one of the major impediments of successful cancer chemotherapy. A large part of resistance of tumors to chemotherapy is caused by the ABC transporter P-glycoprotein encoded by the ABCB1 gene. The main aim of this study was to assess the prognostic value of ABCB1 genotype and phenotype in breast cancer. Methods Six ABCB1 single nucleotide polymorphisms (SNPs) were determined in 90 Czech breast cancer patients by a novel method that allows simultaneous assessment of multiple polymorphisms on a single electronic microarray. Expression levels of ABCB1 were quantified in tumor and nontumor samples of breast cancer patients by real-time PCR. T-test, analysis of variance and Fishers exact test were used to analyze the effect of ABCB1 polymorphisms on ABCB1 expression levels and for the analysis of associations between ABCB1 expression, genotype and clinical and pathological characteristics. Results ABCB1 was expressed in 98.9% of the tumor and in 97.5% of the nontumor samples. ABCB1 was downregulated in 79.5% of tumors compared with the nontumor samples. No significant correlation was observed between ABCB1 mRNA expression levels and clinical and pathological characteristics. High frequencies of the variant alleles in ABCB1 exon 12 (1236C>T, 38.3%) and exon 26 (3435C>T, 54.0%) were observed. Individuals with variant alleles in exons 12 and 26 had significantly lower ABCB1 expression levels in their tumors. SNPs in exons 12 and 26 also correlated with estrogen receptor status of patients. Conclusion ABCB1 SNPs may affect function of P-glycoprotein by influencing the expression level and modify breast cancer prognosis.

Quantitative monitoring of cyclin D1 expression: A molecular marker for minimal residual disease monitoring and a predictor of the disease outcome in patients with mantle cell lymphoma

In mantle cell lymphoma (MCL), minimal residual disease (MRD) is an indicator of the disease outcome. Quantitative methods used so far do not provide a suitable molecular marker in 30–70% patients with MCL (depending on the technique used). We tested cyclin D1 as a marker for quantitative MRD monitoring. The real‐time PCR of cyclin D1 mRNA was performed in 144 bone marrow (BM) specimens including 95 BMs from MCL patients, 39 BMs from patients with other B‐cell non‐Hodgkins lymphomas and 10 BMs from healthy volunteer donors. In 73 BMs obtained from 20 MCL patients we examined the cyclin D1 level during the treatment and follow‐up period. We detected a cyclin D1 overexpression exclusively in BMs infiltrated with MCL, including minimal residual infiltration. Dynamics of cyclin D1 correlated with the patients clinical status in 69/73 BMs. Individual monitoring of patients during the disease course showed cyclin D1 quantitative changes accompanying either the disease relapse or a successful treatment response or the disease‐free survival (remission) and it showed a predictive significance. Cyclin D1 detection is a promising approach for the quantitative MRD monitoring in MCL patients, and the individual monitoring of the cyclin D1 dynamics represents a suitable indicator of the disease course.

Molecular and immunohistochemical analyses of BCL2, KI-67, and cyclin D1 expression in synovial sarcoma

Synovial sarcoma (SS) is a highly aggressive soft tissue sarcoma that causes death in more than half of the affected patients. An immunohistochemical and molecular study of the BCL2, MKI67, and CCND1 genes (expressing the BCL2, KI-67, and cyclin D1 proteins, respectively) was performed to determine the expression profiles in correlation with mRNA levels, and to assess the possible utility of these genes as a potential target for the treatment. Cyclin D1 staining was identified in 18 of 30 cases (60%), and CCND1 mRNA was overexpressed in 15 of 32 cases (47%). KI-67 nuclear immunoreactivity was found in 14 of 29 cases (48%), and MKI67 mRNA was overexpressed in 12 of 32 cases (37.5%). The high level of MKI67 mRNA was observed predominantly in monophasic SS. BCL2, a negative regulator of apoptosis, was expressed in all 32 cases. The intensity of the BCL2 protein expression correlated well with the mRNA level (P<0.0001). The high level of BCL2 mRNA correlated with a high level of CCND1 mRNA, but not with MKI67 mRNA level. Despite advances in therapy of sarcomas, the prognosis of patients with SS remains unfavorable, and a search for an improved therapy approach remains necessary. The strong immunopositivity of BCL2 in SS correlates well with a high level of BCL2 mRNA. Treatment with antisense BCL2 (G3139) may therefore represent an appropriate alternative therapy for patients with BCL2-positive synovial sarcomas.

Quantitative measurement of cyclin D1 mRNA, a potent diagnostic tool to separate mantle cell lymphoma from other B-cell lymphoproliferative disorders.

Cyclin D1 overexpression as a result of t(11;14) is a specific marker for diagnosis of mantle cell lymphoma (MCL) and plays an important role in MCL pathogenesis. To set a highly reliable cutoff value that discriminates MCL from other B-cell lymphoproliferative disorders, we performed a retrospective study of cyclin D1 expression. We established cyclin D1 expression level in 116 frozen and formalin-fixed, paraffin-embedded primary tumors from patients diagnosed with a variety of B-cell lymphoproliferative disorders. We used real time quantitative reverse-transcription polymerase chain reaction to quantify levels of cyclin D1 mRNA. The range of cyclin D1 expression in MCLs exceeded the range found in other lymphomas and reactive lymph nodes by a considerable margin. Cyclin D1 overexpression was found in 60/61 MCLs and in none of the other lymphomas, except for 12/19 mucosa-associated lymphoid tissue lymphomas from the lungs and stomach, which also revealed cyclin D1 overexpression. As epithelial tissues are known to express cyclin D1, an admixture of non-neoplastic epithelial cells present in the extranodal specimens probably influenced the quantitative reverse-transcription polymerase chain reaction result. Quantitative cyclin D1 monitoring provides a diagnostic test and an approach for studying MCL pathogenesis and may be of clinical importance.

Importance of transcript levels of caspase-2 isoforms S and L for breast carcinoma progression

AIM A role of caspase-2 in chemotherapy-induced apoptosis has been suggested. Our study aimed to evaluate the prognostic and predictive importance of caspase-2 isoforms in breast cancer patients. MATERIALS & METHODS Caspase-2L and -2S transcript levels were determined in paired tumor and non-malignant control tissues from 64 patients after neoadjuvant chemotherapy and 100 pretreatment patients (general set) by real-time PCR with absolute quantification. RESULTS Low but statistically significant upregulation of caspase-2L in tumor versus control tissues was observed in both sets. Significant associations of the levels of caspase-2L, -2S or S/L ratio with clinical prognostic factors were observed. However, none of these associations were confirmed in both sets. Levels of caspase-2 isoforms or the S/L ratio did not significantly associate with progression-free survival in the general set or with chemotherapy response in the neoadjuvant set. CONCLUSION Our results suggest that the role of caspase-2 isoforms in the progression of breast cancer may considerably differ between pre- and post-chemotherapy patients.

The role of cytochromes p450 and aldo-keto reductases in prognosis of breast carcinoma patients.

AbstractMetabolism of anticancer drugs affects their antitumor effects. This study has investigated the associations of gene expression of enzymes metabolizing anticancer drugs with therapy response and survival of breast carcinoma patients.Gene expression of 13 aldo-keto reductases (AKRs), carbonyl reductase 1, and 10 cytochromes P450 (CYPs) was assessed using quantitative real-time polymerase chain reaction in tumors and paired adjacent nonneoplastic tissues from 68 posttreatment breast carcinoma patients. Eleven candidate genes were then evaluated in an independent series of 50 pretreatment patients. Protein expression of the most significant genes was confirmed by immunoblotting.AKR1A1 was significantly overexpressed and AKR1C1–4, KCNAB1, CYP2C19, CYP3A4, and CYP3A5 downregulated in tumors compared with control nonneoplastic tissues after correction for multiple testing. Significant association of CYP2B6 transcript levels in tumors with expression of hormonal receptors was found in the posttreatment set and replicated in the pretreatment set of patients. Significantly higher intratumoral levels of AKR1C1, AKR1C2, or CYP2W1 were found in responders to neoadjuvant chemotherapy compared with nonresponders. Patients with high AKR7A3 or CYP2B6 levels in the pretreatment set had significantly longer disease-free survival than patients with low levels. Protein products of AKR1C1, AKR1C2, AKR7A3, CYP3A4, and carbonyl reductase (CBR1) were found in tumors and those of AKR1C1, AKR7A3, and CBR1 correlated with their transcript levels. Small interfering RNA-directed knockdown of AKR1C2 or vector-mediated upregulation of CYP3A4 in MDA-MB-231 model cell line had no effect on cell proliferation after paclitaxel treatment in vitro.Prognostic and predictive roles of drug-metabolizing enzymes strikingly differ between posttreatment and pretreatment breast carcinoma patients. Mechanisms of action of AKR1C2, AKR7A3, CYP2B6, CYP3A4, and CBR1 should continue to be further followed in breast carcinoma patients and models.