Maria M. Sasiadek

Cancer stem cells: the theory and perspectives in cancer therapy

The cancer stem cell theory elucidates not only the issue of tumour initiation and development, tumour’s ability to metastasise and reoccur, but also the ineffectiveness of conventional cancer therapy. This review examines stem cell properties, such as self-renewal, heterogeneity, and resistance to apoptosis. The ‘niche’ hypothesis is presented, and mechanisms of division, differentiation, self-renewal and signalling pathway regulation are explained. Epigenetic alterations and mutations of genes responsible for signal transmission may promote the formation of cancer stem cells. We also present the history of development of the cancer stem cell theory and discuss the experiments that led to the discovery and confirmation of the existence of cancer stem cells. Potential clinical applications are also considered, including therapeutic models aimed at selective elimination of cancer stem cells or induction of their proper differentiation.

1,3-Butadiene and its epoxides induce sister-chromatid exchanges in human lymphocytes in vitro

Sister-chromatid exchanges (SCEs) were induced in human lymphocytes by 1,3-butadiene and its epoxides 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane. After a pulse treatment of 2 h, 1,3-butadiene produced a weak but reproducible increase in SCEs both with and without S9 mix. The response was similar in cultures of whole blood and of isolated lymphocytes. The 2 epoxide metabolites of butadiene, studied in whole-blood lymphocyte cultures without exogenous metabolic activation, were highly active SCE inducers. The lowest effective concentrations of butadiene, monoepoxybutene, and diepoxybutane were 2000 microM, 25 microM and 0.5 microM, respectively. A slight but dose-dependent increase in SCEs was also observed without an exogenous metabolic system after a 48-h treatment with 1,3-butadiene. Already the lowest concentration tested (500 microM) was effective. Again, the response was similar in cultures of whole blood and isolated lymphocytes, suggesting that the lymphocytes are capable of metabolically activating 1,3-butadiene.

Multiple giant cell lesions in patients with Noonan syndrome and cardio-facio-cutaneous syndrome.

Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS) are related developmental disorders caused by mutations in genes encoding various components of the RAS-MAPK signaling cascade. NS is associated with mutations in the genes PTPN11, SOS1, RAF1, or KRAS, whereas CFCS can be caused by mutations in BRAF, MEK1, MEK2, or KRAS. The NS phenotype is rarely accompanied by multiple giant cell lesions (MGCL) of the jaw (Noonan-like/MGCL syndrome (NL/MGCLS)). PTPN11 mutations are the only genetic abnormalities reported so far in some patients with NL/MGCLS and in one individual with LEOPARD syndrome and MGCL. In a cohort of 75 NS patients previously tested negative for mutations in PTPN11 and KRAS, we detected SOS1 mutations in 11 individuals, four of whom had MGCL. To explore further the relevance of aberrant RAS-MAPK signaling in syndromic MGCL, we analyzed the established genes causing CFCS in three subjects with MGCL associated with a phenotype fitting CFCS. Mutations in BRAF or MEK1 were identified in these patients. All mutations detected in these seven patients with syndromic MGCL had previously been described in NS or CFCS without apparent MGCL. This study demonstrates that MGCL may occur in NS and CFCS with various underlying genetic alterations and no obvious genotype–phenotype correlation. This suggests that dysregulation of the RAS-MAPK pathway represents the common and basic molecular event predisposing to giant cell lesion formation in patients with NS and CFCS rather than specific mutation effects.

The CpG Island Methylator Phenotype Correlates with Long-Range Epigenetic Silencing in Colorectal Cancer

The CpG island methylator phenotype (CIMP), characterized by an exceptionally high frequency of methylation of discrete CpG islands, is observed in 18% to 25% of sporadic colorectal cancers. Another hypermethylation pattern found in colorectal cancers, termed long-range epigenetic silencing, is associated with DNA/histone methylation in three distinct gene clusters at chromosome 2q14.2, showing that DNA hypermethylation can span larger chromosomal domains and lead to the silencing of flanking, unmethylated genes. We investigated whether these two phenotypes are interrelated in colorectal cancers. The CIMP status of 148 sporadic colorectal cancers was determined by methylation-specific PCR. We determined the BRAF V600E mutation by mutant allele–specific PCR amplification. The methylation status of the MLH1 gene and of three CpG islands (EN1, SCTR, and INHBB), corresponding to three distinct clusters along 2q14.2, was determined by methylation-specific PCR. The average number of sites showing methylation in CIMP+ tumors was 2.21, compared with 1.22 for CIMP− individuals, and this difference was highly significant (P = 3.6 × 10−8, Mann-Whitney test). Moreover, all CIMP+ tumors showed hypermethylation of at least one of these loci, in contrast to CIMP− tumors, where 18 (16%) samples remained unmethylated. The mean number of simultaneously hypermethylated CpG islands at 2q14.2 differs significantly between CIMP− and CIMP+ tumors, suggesting varying effects of domain silencing in this region. Given that the number of hypermethylated loci at 2q14.2 likely affects the range of silenced flanking genes, high frequency of simultaneous hypermethylation of three CpG islands (EN1, SCTR, and INHBB) may have potential influence on specific characteristics of CIMP+ colorectal cancers. (Mol Cancer Res 2008;6(4):585–91)

Analysis of adaptive response to bleomycin and mitomycin C

Genetic instability resulting from the disturbances in various mechanisms of DNA-repair is the characteristic feature of cancer cells. One of the possibilities to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems. The existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. In this paper we present the study on the AR induced by two chemical mutagens, bleomycin (BLM) and mitomycin C (MMC), which differ in their action on DNA. BLM is a radiomimetic agent causing mainly single-strand breaks (SSB) and double-strand breaks (DSB) and, thus, inducing chromosomal aberrations (CA). MMC is a potent bifunctional mutagen acting as an alkylating agent, causing DNA cross-links and inducing sister chromatid exchanges (SCEs). The protective effect induced by low doses of tested chemicals was analysed in whole blood human lymphocytes using cytogenetic endpoints (CA for BLM and SCE for MMC, respectively) as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. The pre-treatment with a low dose of BLM-induced almost 50% decrease in the frequency of CA induced by challenging dose (CD), while the protective effect of MMC was below 20%. The higher AR induced by BLM may be related to the repair processing of BLM-induced DNA-damages. There was also a variability in ARs among individuals, which may reflect the differences in individual DNA-repair capacity.

Protein tyrosine phosphatase receptor-like genes are frequently hypermethylated in sporadic colorectal cancer

The activity of phosphatases could be influenced by genetic, as well as epigenetic alterations. In our study, we have investigated the methylation status of four PTPRs: PTPRM, PTPRT, PTPRR and PTPRZ1, which were pre-selected using microarray techniques as being alternatively methylated in sporadic colorectal cancer (CRC). The analyses were carried out on 131 surgical specimens obtained from sporadic CRC patients. The methylation status of the four genes was examined using methyl specific PCR (MSP). The analysis of promoter methylation using an Illumina 27K microarray revealed four protein tyrosine phosphatases PTPRM, PTPRT, PTPRR and PTPRZ1 as being hypermethylated with β-value ⩾0.2 and P⩽0.05. Subsequent analysis using MSP confirmed these observations—the frequency of promoter methylation was significantly higher in tumor cells compared with matched normal tissue for each of the analyzed genes. There was no association observed between the methylation status of PTPRs and either CIMP, K-ras (codon 12) and BRAF (exon 15, V600E) mutations or tumor localization (proximal/distal). The results of our study show a statistically significant difference between promoter methylation in cancerous and healthy tissue. This result supports the hypothesis that the PTPR family has an important role in the etiology of CRC.

Detection of viral DNA sequences in sporadic colorectal cancers in relation to CpG island methylation and methylator phenotype

There is evidence that insertion of viral DNA into a mammalian genome can lead to alterations of methylation patterns. The aim of the present study was to examine the presence of DNA sequences of five human DNA viruses (assessed by PCR): JC polyoma virus (JCV), human adenovirus (AdV), Epstein–Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) and human papillomavirus (HPV) in a cohort of 186 sporadic colorectal cancers (CRCs) and related these data with the methylation status of six CpG island methylator phenotype (CIMP)-specific genes (MLH1, CACNA1G, NEUROG1, IGF2, SOCS1, RUNX3) and seven cancer-related genes markers (p16, MINT1, MINT2, MINT31, EN1, SCTR and INHBB) assessed by methylation-specific PCR in 186 and 134 CRC cases, respectively. The AdV, KSHV and HPV were detected in four (2%), two (1%) and zero CRC cases, respectively, and thus were excluded from further analyses. Although 19% and 9% of the CRCs were positive for EBV and JCV, respectively, no associations between virus presence and CpG island methylation were found after correction for multiple testing. Our results demonstrate that the presence of DNA sequences of JCV and EBV in CRC is unrelated to the methylation of the 13 cancer-related CpG islands and CIMP.

The influence of GSTM1 and GSTT1 genotypes on the induction of sister chromatid exchanges and chromosome aberrations by 1,2:3,4-diepoxybutane

Cytogenetic tests - chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) - are most often applied in biomonitoring of the genotoxicity of potentially carcinogenic chemicals in human cells. One of the extensively studied genotoxins is diepoxybutane (DEB) - reactive biometabolite of butadiene (BD). Several studies showed a high SCE induction in human lymphocytes exposed in vitro to various concentrations of DEB. DEB also proved to be a potent inducer of chromosome aberrations and micronuclei. A bimodal distribution of SCE frequency after in vitro DEB treatment was observed. The aim of the present study was to examine the ability of DEB to induce different individual cytogenetic response measured by SCE and CA frequency. The possible influence of genetic polymorphism has also been taken into account, by including donors representing positive or null GSTM1 and GSTT1 genotypes. Our study supported the earlier results showing that DEB is an effective inducer of SCEs and CAs, causing also the decrease in replication index (RI). DEB bioactivity measured by SCE induction - but not by CA test - was significantly higher in GSTT1 negative than in GSTT1 positive donors. GSTM1 polymorphism had no influence on these endpoints. The donors GSTT1-/GSTM1+ were shown to be slightly more sensitive to DEB than GSTT1-/GSTM1- individuals. There was also observed a unimodal distribution of DEB-induced SCEs and CAs in the group, despite the fact that the experiment was performed on the lymphocytes obtained from both GSTT1 positive and negative donors.

Intermediate- and Low-Methylation Epigenotypes Do Not Correspond to CpG Island Methylator Phenotype (Low and -Zero) in Colorectal Cancer

Background: Most recent genome-wide studies on the CpG island methylation in colorectal cancer (CRC) have led to the discovery of at least 3 distinct methylation clusters. However, there remains an uncertainty whether the CRC clusters identified in these studies represent compatible phenotypes. Methods: We carried out comprehensive genome-scale DNA methylation profiling by Illumina Infinium HumanMethylation27 of 21 DNA pools that represent 84 CRC samples divided according to their high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME, respectively) and 70 normal-adjacent colonic tissues. We have also examined the relationship among 3 epigenotypes and chromosomal gains and deletions (assessed by Comparative Genomic Hybridization) in a group of 100 CRC samples. Results: The HME subgroup showed features associated with CpG island methylator phenotype – high (CIMP-high) including methylation of specific CpG sites (CpGs) as well as significantly lower mean number of chromosomal imbalances when compared with other epigenotypes. The IME subgroup displayed the lowest number of methylated CpGs (717 vs. 2,399 and 2,679 in HME and LME, respectively) and highest mean number of chromosomal imbalances when compared with HME (P, 0.001) and LME (P, 0.004). A comparison between the methylation profiles of 3 epigenotypes revealed more similarities between the HME and LME (1,669 methylated CpGs overlapped) than HME and IME (673 methylated CpGs overlapped). Conclusion: Our results provide evidence that IME and LME CRCs show opposite features to those that have been previously attributed to CIMP-low and CIMP-0 CRCs. Impact: These discrepancies should be considered when interpreting the data from a particular epigenotyping method. Cancer Epidemiol Biomarkers Prev; 22(2); 201–8. ©2012 AACR.

Chromosome aberrations and gene mutations in patients with esophageal atresia.

Esophageal atresia (EA) is one of the most frequent congenital malformations of the gastrointestinal tract. Many genetic alterations in patients with EA have been described in the literature. It is thought that the etiology of EA is heterogeneous. This review of the literature provides detailed information about chromosomal aberrations, gene mutations, and clinical features of neonates with EA, and serves as an excellent source to compare newly diagnosed patients with those described in the literature.