Biomedical subjects

A Försti

Publications and source records attributed to A Försti.

At least 19 recordsLinked to original sources

PubMed2006

Case-control study in basal cell carcinoma of the skin: single nucleotide polymorphisms in three interleukin promoters pre-analysed in pooled DNA.

BACKGROUND: Basal cell carcinoma (BCC) is one of the most common neoplasms in the world. Development of BCC is associated with environmental factors (especially sun exposure) as well as heritable factors. OBJECTIVES: To analyse three single nucleotide polymorphisms (SNPs) in the promoter regions of interleukin (IL) genes in genomic DNA from 527 cases of BCC and 530 matched controls and to examine if DNA pooling is a useful method on which to base decisions regarding further SNP analysis. METHODS: The SNPs analysed were IL6-597, IL10-1082 and IL1B-511. The SNPs were first analysed from pooled DNA and afterwards from individual samples. The DNA pools resulted from a division of the samples into cases and controls, female and male, and three age groups. In these pools the allele frequencies were estimated by two methods, real-time polymerase chain reaction with allele-specific primers, and quantitative sequencing. RESULTS: No significant association was found when the allele frequencies in cases and controls were compared. However, by analysis of the individual genotypes we found SNP IL6-597 G/A to be significantly associated with BCC risk (P =0.007). Hereby the heterozygous genotype 'GA' had a protective effect (odds ratio 0.64, 95% confidence interval 0.49-0.84). No significant association was found for IL10-1082 and IL1B-511. CONCLUSIONS: The association of SNP IL6-597 with BCC could be found only by individual genotyping, but would have been missed if only data from the pooling analysis had been known.

Aged↗

PubMed2005

Association of polymorphisms and haplotypes in the human growth hormone 1 (GH1) gene with breast cancer.

The growth hormone 1 (GH1)/insulin-like growth factor I (IGF-I) axis plays an important role in the development of breast cancer. By binding to its receptor, GH1 stimulates the production of IGF-I and its binding protein IGFBP3, resulting in the regulation of cell proliferation, differentiation and apoptosis. The GH1 gene expression is regulated by a highly polymorphic proximal promoter and a distal locus control region (LCR) 14.5 kb upstream of the gene. We investigated the effect of single nucleotide polymorphisms (SNPs) in the LCR and in the promoter region and an intron 4 SNP (IVS4+90 T/A) on breast cancer risk in a large cohort of Polish and German familial breast cancer cases and controls. SNPs in the LCR did not show an influence on breast cancer risk, either alone or in haplotypes. Three SNPs in the promoter region (G-340T, A-68G/C and A-63T/C) showed an increased and four SNPs (A-137G, G-119T, G-93delG and T-4G) a decreased allele frequency in the cases compared with the controls. Two of the SNPs (A-137G and G-93delG) lead to a decreased breast cancer risk among the minor allele carriers in the joint analysis of the two populations (odds ratio (OR) 0.62, 95% confidence interval (95% CI) 0.44-0.89, P = 0.01 and OR 0.65, 95% CI 0.47-0.90, P = 0.01, respectively). Haplotype analysis with these seven promoter SNPs revealed a protective association (OR 0.61, 95% CI 0.37-1.00, P = 0.04) for the haplotype GAGdAAT, containing the G-93delG variant allele, which in the single analysis already showed a protective effect. The effect was marginally stronger in combination with the LCR GC haplotype (OR 0.49, 95% CI 0.23-1.01, P = 0.04).

Adult↗

PubMed2002

Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours.

In this study we screened the histone acetyltransferases CBP and PCAF for mutations in human epithelial cancer cell lines and primary tumours. We identified two CBP truncations (both in cell lines), seven PCAF missense variants and four CBP intronic microdeletions. These data suggest that neither gene is commonly inactivated in human epithelial cancers.

Acetyltransferases↗

PubMed2001

Loss of heterozygosity in tumour-adjacent normal tissue of breast and bladder cancer.

Normal tumour-adjacent breast tissue samples from 12 breast cancer patients forming six monozygotic twin pairs were analysed for loss of heterozygosity (LOH) on chromosomes 1, 13 and 17. 7 patients showed LOH at one or more markers. Each of them had a different LOH pattern. Only one twin pair showed LOH at the same locus, but the twins had lost a different allele. Multiple (n=1-13), histologically normal samples were collected from 6 bladder cancer patients and analysed for LOH on chromosomes 3 and 9. On chromosome 9, all 6 patients analysed showed LOH in at least one sample and one marker. Four of them also showed LOH on chromosome 3. Samples surrounding different tumours of a given patient resembled each other. More heterogeneity was seen between the patients, even though they shared some similarities in LOH clustering. The results demonstrate that tumour-adjacent normal tissues already harbour genetic changes typical for tumours. These alterations can reveal the earliest changes leading to tumorigenesis.

Breast Neoplasms↗

PubMed2001

Allelic imbalance on chromosomes 13 and 17 and mutation analysis of BRCA1 and BRCA2 genes in monozygotic twins concordant for breast cancer.

To study genetic changes associated with the development of breast cancer and the extent of its hereditary predisposition, paraffin-embedded tissue samples were obtained from monozygotic twin pairs concordant for breast cancer through the linked Swedish Twin and Cancer Registries. DNA samples extracted from the matched tumour and normal tissues of nine twin pairs were analysed for allelic imbalance using a series of microsatellite markers on chromosomes 13 and 17, containing loci with known tumour suppressor genes. Multiple losses of constitutional heterozygosity (LOH), consistent with a loss of large genomic region, the whole chromosome or chromosome arm, was found in at least three pairs of twins. One double mitotic crossover was identified in one tumour sample in a pair concordant for LOH at multiple loci on both chromosomes. Recombination breakpoints were mapped to regions delineated by D13S218 and D13S263, and D13S155 and D13S279, respectively. In general, no genetic effect of losing the same allele within a twin pair was found. However, for one marker at chromosome 13 (D13S328, between the BRCA2 and the RB-1 loci) and two markers on chromosome 17 (D17S786, distal to the p53 locus, and D17S855, an intragenic BRCA1 marker) the proportion of twin pairs with the same LOH was significantly higher than expected. These regions may reflect hereditary genomic changes in our sample set. In addition, tumour DNA samples from a subset of 12 twin pairs were analysed for BRCA1 and BRCA2 mutations using exon-by-exon single-strand conformation polymorphism analysis. Two unclassified BRCA2 variants, with a putative pathogenic effect, were identified, but no pathogenic alterations were found in the BRCA1 gene.

Adult↗

PubMed2001

Use of monozygotic twins in search for breast cancer susceptibility loci.

We have used Swedish monozygotic twins concordant for breast cancer to study genetic changes associated with the development of breast cancer. Because loss of heterozygosity (LOH) at a specific genomic region may reflect the presence of a tumour suppressor gene, loss of the same allele in both of the twins concordant for breast cancer may pinpoint a tumour suppressor gene that confers a strong predisposition to breast cancer. DNA samples extracted from the matched tumour and normal tissues of nine twin pairs were analysed for allelic imbalance using a set of microsatellite markers on chromosomes 1, 13, 16 and 17, containing loci with known tumour suppressor genes. The two main regions, where more twin pairs than expected had lost the same allele, were located at 16qtel', including markers D16S393, D16S305 and D16S413, and at 17p13, distal to the p53 locus. Our results show that the monozygotic twin model can be used to suggest candidate regions of potential tumour suppressor genes, even with a limited number of twin pairs.

Adult↗

PubMed2000

Single nucleotide polymorphism analyses of the human proliferating cell nuclear antigen (pCNA) and flap endonuclease (FEN1) genes.

The products of proliferating cell nuclear antigen (PCNA) and flap endonuclease (FEN1) genes are multifunctional proteins that are involved in DNA replication and damage repair. Yeast models suggest association of mutant forms of PCNA and FEN1 with genomic instability. In our study, we have determined the single nucleotide polymorphisms in human PCNA and FEN1 genes. We sequenced the coding region and adjacent noncoding region of both the PCNA and FEN1 genes in 120 alleles (60 individuals). In the PCNA gene, we detected 9 sequence variants with Hardy-Weinberg allele frequency ranging from 0.008 to 0.088. No polymorphism was detected in the FEN1 gene. The sequence variants in the PCNA gene included 7 intronic single nucleotide polymorphisms (SNP) and 2 synonymous exonic SNPs. All the intronic SNPs were located in introns 1 and 4, which contain several regulatory elements involved in the control of PCNA gene expression. Six of the 7 intronic SNPs showed complete linkage disequilibrium. We confirmed this allelic linkage disequilibrium by allele-specific PCR sequencing. We genotyped 117 additional individuals belonging to 3 population subgroups using the PCR-RFLP method. Finally, to see if the detected polymorphisms are associated with any cancer type, we genotyped cases with melanomas (37 cases), breast cancers (118 cases) and lung cancers (100 cases). We did not find statistical difference in frequency of polymorphism in any cancer type compared with healthy controls, although in breast cancer the frequency was low. Our results suggest that the coding regions of the PCNA and FEN1 genes are highly conserved when compared with other DNA repair genes. The potential of some of the detected intronic polymorphisms to effect regulation of the PCNA gene expression remains to be determined.

Alleles↗

PubMed1999

DNA copy number losses at 1p32-pter in monozygotic twins concordant for breast cancer.

To find similarities that may possibly indicate novel mutations, we performed comparative genomic hybridization (CGH) analysis following degenerate oligonucleotide primed polymerase chain reaction (PCR) for DNA obtained from unique material of breast cancer that developed in monozygotic twin-pairs. Polymerase chain reaction amplification was successful in 12 samples for 11 patients, including 3 pairs. Six samples exhibited DNA copy number changes. Gains (76%) were more frequent than losses (24%). Gains or high-level amplifications in 8q were present in all but 1 of the abnormal cases. Frequent gains were detected with a minimal common overlapping region at 5p (4 cases), at 1q25-qter (3 cases), and at 20q12-qter (2 cases). The most frequent loss, detected in half of the abnormal cases, was at 1p32-pter. One twin-pair showed similar changes in 4 chromosomal locations involving loss of 1p32-pter and gains in 1q25-qter, 5, and 8q.

Base Sequence↗

PubMed1997

Use of twins in search for tumor suppressor genes.

A new approach is applied in the mapping of tumor suppressor genes: analysis of loss of heterozygosity (LOH) in concordant tumors of monozygotic and dizygotic twins. The method relies on recognition of genome locations undergoing loss in both twins in a high proportion of the set of all twin pairs examined. The method effectively pinpoints, and excludes, the loci of potential tumor suppressor genes. With the help of a high density linkage map any such candidates can be placed within a narrow region of a chromosome arm and perhaps matched with known genes. The analysis of the Swedish Twin Registry has shown a clear genetic component for breast cancer. We have identified mono- and dizygotic twins concordant for breast cancer and collected the pathology specimens. Tumor and normal tissue was microdissected and microsatellite analysis carried out to test for allelic loss (LOH) in entirely new and putative chromosomal loci in this cancer. It can be calculated that using only six pairs of informative monozygotic twins, a locus can be incriminated with a high probability. Using increasingly dense markers and search for homozygous deletions, it should be possible to map one or more candidates for breast cancer.

BRCA1 Protein↗

PubMed1995

Analysis of UV-induced DNA photoproducts by 32P-postlabelling.

UV-induced cyclobutane dimers and 6-4 photoproducts, containing an unmodified nucleotide at the 5'-position were released from DNA by means of digestion with DNase I, snake venom phosphodiesterase and prostatic acid phosphatase. The enzymes were deactivated by proteinase K followed by ethanol precipitation. The products were phosphorylated by polynucleotide kinase and [gamma-32P]ATP. The TLC system used for the analysis enables separation of the different photoproducts and detection at a fmol level. T4 endonuclease treatment was applied to confirm the positions of cyclobutane dimers.

Animals↗

PubMed1995

32P-postlabelling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA.

A 32P-postlabelling method is reported for the detection of 7-alkylguanines, the major adducts formed by the reaction of 1,2-alkylepoxides with DNA. Calf thymus DNA was reacted in vitro with different epoxides (ethylene oxide through octylene oxide) and digested with micrococcal nuclease and spleen phosphodiesterase to 3'-nucleotides. The adduct enrichment was carried out by an ion-exchange method and adducts were labelled with [gamma-32]ATP in a T4-polynucleotide kinase-mediated reaction. The labelled adducts, prior to resolution by two-dimensional TLC, were treated with nuclease P1. The recoveries of adducts formed by different epoxides ranged from 3 to 10%, tending to increase with the increase in the chain length of the substitutions. The 32P-postlabelled adducts were also analysed by HPLC coupled with a radioisotope detector. This method has been applied for the detection of 7-alkylguanine adducts in rats exposed to different alkenes. The method has potential for use in measuring human exposure to alkenes or their corresponding epoxides as well as the endogenously formed 7-alkylguanine adducts.

Animals↗

PubMed1994

A 32P-postlabelling assay for DNA adducts induced by cis-diamminedichloroplatinum(II).

A 32P-postlabelling method was developed to measure cisplatin-DNA adducts. Platinated oligonucleotides with different chain lengths were enzymatically digested with deoxyribonuclease I, snake venom phosphodiesterase (SVPD) and prostatic acid phosphatase. We found that SVPD was not able to cut the phosphodiester bond immediately 5' to the platinated nucleotide. As a result the adducts had an attached 5' unmodified nucleotide, while the unmodified nucleotides were digested to nucleosides. This is a facile enrichment procedure for the adducts, because the normal nucleosides lacking the 3'-phosphate are not substrates for T4 polynucleotide kinase. Instead, the adduct fragments containing an unmodified nucleotide at their 5' end can be phosphorylated by T4 polynucleotide kinase and [-32P]ATP. This method was also shown to be suitable for the detection of cisplatin-adducts in platinated calf thymus DNA.

Chromatography, High Pressure Liquid↗

PubMed1994

Comparison of TLC- and HPLC-32P-postlabelling assay for cisplatin-DNA adducts.

Calf thymus DNA, treated with the antitumour agent cis-diamminedichloroplatinum(II) (cisplatin), was enzymatically digested with deoxyribonuclease I, snake venom phosphodiesterase and prostatic acid phosphatase. As a result the adducts were released with an unmodified nucleotide at their 5'-side. The adducts were postlabelled with [gamma-32P]ATP and separated by thin-layer chromatography (TLC) and HPLC with on-line detection of 32P. One of the standards, platinated d(TGG), could be analysed with a recovery of 31% with both the TLC and the HPLC separation systems, when the amount of adduct was between 0.5 and 100 fmol. In the case of platinated DNA the linear part of the concentration curve was from 100 fmol down to the lowest amount of adducts measured, 3.2 fmol, with the TLC system and between 8 and 40 fmol with the HPLC system. The recoveries of the adducts were 28% and 16% respectively.

Acid Phosphatase↗

PubMed1993

Development of a 32P-postlabeling assay for 7-methylguanines in human DNA.

The application of a 32P-postlabeling assay for 7-methylguanines in DNA was studied either by labeling the imidazole ring-opened dinucleotide derivatives or by using strong-anion-exchange column chromatography for the adduct enrichment from normal nucleotides. Data showed that 7-methylguanines can be efficiently labeled as dinucleotides when in vitro methylated DNA was first imidazole ring-opened and then digested to the dinucleotide level with deoxyribonuclease I, snake venom phosphodiesterase, and prostatic acid phosphatase. When using ion exchange chromatography for the adduct enrichment, DNA was digested with micrococcal nuclease and spleen phosphodiesterase. Anion exchange chromatography was applied for 7-methylguanine measurements in white blood cell DNA of healthy nonsmokers (n = 17) and patients (n = 4) treated with the methylating drugs procarbazine and decarbazine. We found that the mean level of 7-methylguanine residues in nonsmokers was 2.5 per 10(7) nucleotides. The corresponding level in the patient samples immediately after the drug treatment was 57 per 10(7) nucleotides.

Adult↗

PubMed1993

Testing of quantitative parameters in the 32P-postlabelling method.

The 32P-postlabelling technique involves many steps that need to be carefully controlled in order to obtain a reliable quantitative determination of DNA adducts. We have studied several of the parameters involved in the DNA digestion procedures as well as those concerned in the phosphorylation reaction. Since adducts behave in very different ways in the labelling reaction, an individual protocol has to be worked out for each particular type of adduct. Quantitation is usually possible only if a synthesized standard of the adduct under investigation is run in parallel to the DNA samples throughout the whole procedure.

Adenosine Triphosphate↗

PubMed1991

Measurement by 32P-postlabelling of 7-methylguanine levels in white blood cell DNA of healthy individuals and cancer patients treated with dacarbazine and procarbazine. Human data and method development for 7-alkylguanines.

A 32P-postlabelling method was developed to measure 7-methylguanine in human DNA. DNA was digested to nucleotides and 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-me-dGMP) was isolated from normal nucleotides using strong anion-exchange column chromatography. Overall the method gave 35-45% yield as measured with DNA methylated with tritiated dimethyl sulfate. Total white blood cell DNA from healthy non-smokers (n = 17) contained from 2.5 7-methylguanine residues/10(7) nucleotides, corrected for the losses in preparation. Among four patients sampled immediately after a total dose of 1050-2800 mg of dacarbazine or procarbazine, the mean adduct level was 57 7-methylguanine residues/10(7) nucleotides. As further method development, we also investigated the phosphorylation reaction by T4 polynucleotide kinase using dinucleotides containing 7-methylguanine and corresponding imidazole ring-opened products as substrates. We found that imidazole ring-opened dTpdG-Me is resistant to digestion with deoxyribonuclease I, snake venom phosphodiesterase and prostatic acid phosphatase. It is quantitatively phosphorylated at femtomolar levels. This method is shown to be suitable for the detection of 7-methylguanine in DNA, and is suggested to be the approach most suited to postlabelling large and labile 7-alkylguanines in DNA.

Chromatography, Thin Layer↗

PubMed1990

The influence of N-7 platination and methylation on the stability of deoxyguanosine and deoxyguanylyl-(3'-5')-deoxyguanosine.

The rates of degradation of N-7 platinated deoxyguanosine (dG) and deoxyguanylyl-(3'-5')-deoxyguanosine (dGpdG) were followed in 66 mM Tris-HCl (pH 7.4) at 100 degrees C. The half-life for the intramolecular cross-link of cis-diamminedichloroplatinum(II) (cis-Pt) between two neighboring guanines (Pt-dGpdG) was 86 min, and the half-life of the intermolecular cross-link of cis-Pt on two guanines (dG-Pt-dG) was 54 min. For comparison the half-lives of dGpdG and the N-7 methylated dGpdG (Me-dGpdG) were 636 min and 11 min, respectively. The end product of the degradation of dGpdG, Pt-dGpdG and dG-Pt-dG was guanine, while Me-dGpdG was degraded to 7-methylguanine. In the case of dG-Pt-dG the main reaction pathway was through the depurination of one of the deoxyguanosines; in the case of Pt-dGpdG the degradation occurred either through the cleavage of one of the N-glycosidic bonds or through the cleavage of one of the Pt-bonds.

Chromatography, High Pressure Liquid↗

PubMed1989

Kinetics of reaction of cis-diamminedichloroplatinum(II) with DNA.

A method based on cation exchange chromatography was developed to determine the adducts formed in the reaction of cis-diamminedichloroplatinum(II) (cis-Pt) with DNA. DNA was incubated with various concentrations of cis-Pt for various periods of time, ethanol precipitated, and enzymatically digested to nucleosides and Pt-containing oligonucleotides. The unmodified nucleosides were separated from the positively charged intra- and interstrand cis Pt adducts with a weak cation exchanger, CM-Sephadex C-25, and the adducts were further purified by HPLC. The main adduct was shown to be an intrastrand cross-link of cis-Pt bound to the N-7 atoms of two neighboring guanines. The minor adducts were intra- and interstrand cross-links of cis-Pt with adenine and guanine and an interstrand cross-link of cis-Pt with two guanines. At low levels of DNA-modification (cis-Pt:nucleotide = 1:50-1:1000) the intrastrand cross-link of cis-Pt with two guanines consisted of 60-70% of the total platination of DNA. At higher levels of DNA-modification (greater than 1:20), the amount of undigested products increased, indicating shielding of DNA by cis-Pt from nucleolytic enzymes.

Chromatography, High Pressure Liquid↗