Biomedical subjects

R A Lothe

Publications and source records attributed to R A Lothe.

68 records · Page 4Linked to original sources

PubMed1992

Molecular genetic studies of tumor suppressor gene regions on chromosomes 13 and 17 in colorectal tumors.

BACKGROUND: In the majority of colorectal carcinomas, both copies of the tumor suppressor gene TP53 (tumor protein 53) are known to be inactivated. In contrast to a loss of tumor suppressor function, it has been suggested that an increased copy number of the RB1 gene is involved in the progression of these tumors. PURPOSE: To determine genetic alterations at chromosomes 13 and 17 in colorectal tumors, we have studied several loci on these chromosomes, with special focus on the RB1 and TP53 genes at both the level of DNA sequence and the level of gene expression. METHODS: Restriction fragment length polymorphism analysis was performed after alkaline Southern blotting of the DNA fragments and hybridization (in 7% sodium dodecyl sulfate and 0.5 M NaPO4) of the nylon membranes with multiprimed, radioactively labeled probes. Total RNA was extracted from tissue biopsy specimens by homogenization of the samples in guanidinium thiocyanate followed by separation in a CsCl gradient. By use of an image-processing system, x-ray film signals were measured densitometrically. Point mutations within the TP53 gene were detected by use of polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis. Direct sequencing of PCR products revealed the exact nature of the mutations. Protein expression of TP53 was seen by immunostaining of sections from paraffin-embedded material using a mouse monoclonal antibody. The two-sided Fisher's Exact Test was used for statistical analysis. RESULTS: An increase in allelic copy number at 13q loci was seen in 10 (32%) of 31 tumors. In the majority of the cases, this increase probably reflected a change in the diploid status of chromosome 13; in some cases, however, only part of the 13q seemed to be involved. The RB1 gene showed an elevated level of RNA compared with the beta-actin signal. Fourteen (48%) of 29 tumors showed loss of heterozygosity at loci on 17p, and base mutations within the TP53 gene were seen in 14 (42%) of 33 tumors. RNA and protein analyses of TP53 revealed an increased level of expression in the tumors compared with normal mucosa. Allelic variations seen at 13q and 17p were not associated (P = .7). CONCLUSIONS: Our results suggest that, in addition to aneuploidy, gain of specific chromosome 13 sequences is involved in the tumorigenesis of the colon and rectum. In addition, they confirm the importance of TP53 mutations for the progression of such tumors and support the view that accumulation of events is more important than the order of events. The genetic changes observed at chromosome arms 13q and 17p seem to be independent of each other.

Adolescent↗

PubMed1992

High resolution chromosome banding in search of germ line mutations applied on testicular cancer patients.

Patients with bilateral and/or familial testicular cancer are assumed to demonstrate germ line mutations if such mutations are instrumental in testicular cancer patients. Constitutional rearrangements have to our knowledge not been reported, while an increased level of constitutional chromosome instability has been claimed. Lymphocytes from 12 Norwegian patients with bilateral or familial testicular cancer have been studied by high resolution Giemsa banding. A possible germ line mutation would be expected to be seen in every cell. It is therefore sufficient to examine one pair of the individual chromosomes obtained, if necessary, from several cells. Based on our previous findings of loss of heterozygosity in the chromosome regions 3p and 11p in testicular tumors, these chromosome arms were paid special attention. However, no mutations were observed in these regions nor in any other chromosome. Applying a 95% confidence interval, we conclude that less than 23% of familial and/or bilateral testicular cancer patients are caused by germ line mutations large enough to be detected with high resolution banding of at least 450 G-bands per haploid genome. The presented analyses were used to standardize the resolution determination in our laboratory.

Adolescent↗

PubMed1992

Chromosome 12 in human testicular cancer: dosage changes and their parental origin.

Cytogenetically, a marker chromosome interpreted as i(12p) is present in most testicular tumors of germ cell origin. In this study, 22 patients with testicular germ-cell tumors were investigated by Southern blot hybridization to characterize changes in chromosome 12. In comparison with normal DNA, tumor DNA of 18 patients showed increased dosages of 12p accompanied by a comparable or smaller increase or no change in the dosage of centromeric sequences of chromosome 12. A likely interpretation was that most testicular tumors had one or several isochromosomes for 12p that were formed by somatic division of the centromere and that the points of breakage and reunion in the centromeric region were different in different tumors. Allelic 12p fragments showing increased intensity were paternal in four and maternal in three of seven informative cases. Thus, there was no evidence of sex-limited parental imprinting. Furthermore, the observed patterns of allelic fragments suggested that the marker was an i(12p) formed by sister chromatids of one homolog number 12 rather than the result of interchange of genetic material between different homologues.

Alleles↗

PubMed1991

Genetic alterations within the retinoblastoma locus in colorectal carcinomas. Relation to DNA ploidy pattern studied by flow cytometric analysis.

Alterations within the retinoblastoma (Rb) gene, as detected by the VNTR probe p68RS2.0, and flow cytometric DNA pattern have been analysed in 255 colorectal carcinomas. A total of 35.3% of the tumours had alterations within the Rb gene. Amplification of one allele was demonstrated in 29.5% of the tumours, and loss of heterozygosity was found in 11.5%. No association was found between amplification within the Rb gene and clinicopathological characteristics of the patients. The high frequency of alterations demonstrated within the Rb gene, suggests that this gene is involved in colorectal carcinogenesis with amplification as by far the most abundant genetic alteration. This may imply that the Rb gene has an oncogene-like function in colorectal carcinomas, rather than acting as a tumour suppressor gene. Sixty-three per cent of the carcinomas were DNA aneuploid, and a significant association was demonstrated between amplification within the Rb gene and DNA aneuploidy (P less than 0.01). Two other chromosome loci were analysed, on chromosome 1p (probe pYNZ2) and on chromosome 2p (probe pYNH24), respectively. On chromosome 1p, heterozygous loss was found in 22.2% of the tumours, indicating an involvement of this chromosome in a subset of colorectal carcinomas.

Alleles↗

PubMed1990

High-resolution karyotypes of eighteen Norwegian polyposis patients.

High-resolution karyotypes were analyzed from lymphocyte cultures of 18 Norwegian polyposis patients, of whom two had classic Gardner syndrome (GS). We focused on possible rearrangements of the long arm of chromosome 5. Previously the gene for familial adenomatous polyposis (FAP) was localized to the region 5q21-22 by linkage analysis. A patient with a constitutional deletion in this region would be of great value in the course of cloning the gene. At the level of high-resolution G-banding, however, none of these patients showed any interstitial deletion or other rearrangement on chromosome 5 or any other chromosome.

Adenomatous Polyposis Coli↗

PubMed1990

Human pepsinogen A (PGA): an informative gene complex located at 11q13.

Human pepsinogen (PGA) exhibits extensive polymorphism that can be detected both at the protein and the DNA level. We describe here two restriction fragment length polymorphisms, EcoRI and BglII, which provide for the detection of three of the most common PGA haplotypes (A, B, and C) in the United States population. The relationship of these polymorphisms to each PGA haplotype was determined by analysis of DNA from individuals exhibiting the corresponding protein phenotypes and by analysis of a series of human x mouse somatic cell hybrids containing the individual chromosome 11 homologous from heterozygous individuals exhibiting the AB and AC protein phenotypes. The use of the BglII polymorphism in combination with previously described EcoRI polymorphism provides a very informative marker of 11q13.

Animals↗

PubMed1989

Loss of 3p or 11p alleles is associated with testicular cancer tumors.

Constitutional and tumor genotypes defined by polymorphic DNA markers were examined in 31 testicular cancer patients. Constitutional karyotypes were analyzed and clinical data presented. We analyzed 11 loci representing 8 chromosomes, including regions frequently deleted in other types of cancer. Loss of 3p or 11p sequences was detected in 8 of 28 heterozygotes (28%) and in 5 of 20 heterozygotes (25%). This gives a combined total loss of 40%. The other autosomal loci tested showed no loss or a loss of less than 10% of alleles. We suggest that this loss of heterozygosity for genetic material on chromosome 3p or on 11p is nonrandom and important in the development of a major subset of testicular neoplasms.

Adult↗

PubMed1988

VNTR (variable number of tandem repeats) markers show loss of chromosome 17p sequences in human colorectal carcinomas.

Restriction fragment length polymorphisms at 53 autosomal loci were screened for heterozygosity in 40 colorectal cancer patients. The DNA pattern in constitutional versus tumor/polyp tissue was compared. More than half of the markers tested were of the VNTR (variable number of tandem repeats) type, giving the patient panel a higher informational content, since the frequency of individuals heterozygous for a particular marker is increased. Loss of alleles was revealed in 40% of the tumors from constitutionally heterozygous patients at the chromosome 17p loci, identified by the markers YNH37 and YNZ22. Similar losses were also detected on other autosomes, but at a significantly lower frequency. Our results suggest that hemi/homozygosity of 17p alleles plays a role in the development of a major subset of colorectal carcinomas. Similar observations regarding other autosomal loci may be interpreted as random losses in these tumors, or they may indicate loci important to minor clinical subclasses of colon carcinomas.

Carcinoma↗

PubMed1987

Loss of one chromosome #13 during development of a polyposis tumor.

A cell panel from six different familial cancers, where both normal and tumor tissues were available, was examined for genotypic changes with polymorphic DNA probes. Seventeen probes were tested, representing chromosomes #1, #2, #5, #6, #13, #14, #17, and #19. One probe, p7F12 (D13S1, at 13q12-q14) revealed loss of heterozygosity in two tumors: an osteosarcoma from a patient with retinoblastoma that had been included as a control, and one polyposis tumor that had been established in nude mice from a duodenal carcinoma biopsy. Loss of heterozygosity was observed in the first passage of the mouse tumor. Chromosome analysis in later passages revealed loss of one whole chromosome #13 as the single consistent karyotypic change.

Adenomatous Polyposis Coli↗

PubMed1986

Very close linkage between D2S1 and ACP1 on chromosome 2p.

The genomic DNA-probe L2.30 was used to assign D2S1 to 2p23-pter by in situ hybridization. The RFLP revealed by BglII was then used for linkage studies in the Oslo-NHIK families segregating for the acid phosphatase ACP1 protein polymorphism. Evidence for very close linkage was found by a lod score of +17.17 at recombination fraction theta = 0.01. By this close linkage 92 informative meioses could be inferred from the families and with only a single crossover. The upper probability limit to the recombination fraction is 0.06 according to the HGM 8 criterion. No association between ACP1 alleles and D2S1 BglII alleles was found. The Norwegian gene frequencies for D2S1 were A1 (9.0 kb) = 0.65 and A2 (6.3 kb) = 0.35.

Acid Phosphatase↗

PubMed1985

Stainer and Scholte's pertussis medium with an alternative buffer.

A comparative study of the Stainer and Scholte chemically defined growth medium (SS) for Bordetella pertussis, and a modification of it (MSS), was made. The Tris buffer which is reported to have adverse effects was replaced in MSS with sodium glycerophosphate. By using generation time as a measure of the growth promoting properties of the media, we found that the MSS was superior to SS for B. pertussis strain CN5612/67, which was used as a vaccine strain in Norway. Additionally by employing the 'dilution to extinction' method we showed that MSS supported B. pertussis growth from smaller inocula than the original SS medium. No significant differences in the quality of vaccines made from organisms grown in the two alternative media were observed in animal tests for potency and safety. The difference in buffers in SS and MSS had no influence on the formation of the agglutinogens, as tested by agglutination with specific factor sera. It was confirmed that liquid media gave a better total expression of the agglutinogens than solid media.

Agglutination↗

PubMedDate not supplied

Evaluation of 1p losses in primary carcinomas, local recurrences and peripheral metastases from colorectal cancer patients.

Cytogenetic and molecular genetic analyses of colorectal adenomas and carcinomas have shown that loss of the distal part of chromosome arm 1p is common, particularly in tumors of the left colon. Because the importance of 1p loss in colorectal cancer metastases is unknown, we compared the frequency, exact site and extent of 1p deletions in primary carcinomas (n=28), local recurrences (n=19) and metastases (n=33) from 67 colorectal cancer patients using 14 markers in an allelic imbalance study. Loss of 1p was found in 50% of the primary carcinomas, 33% of the local recurrences, and 64% of the metastases, revealing a significant difference between the local recurrences and the metastases (P=.04). The smallest region of 1p deletion overlap (SRO) defined separately for each group of lesions had the region between markers D1S2647 and D1S2644, at 1p35-36, in common. The genes PLA2G2A (1p35.1-36) and TP73 (1p36.3) were shown to lie outside this consistently lost region, suggesting that neither of them are targets for the 1p loss. In the second part of the study, microdissected primary carcinomas and distant metastases from the same colorectal cancer patients (n=18) were analyzed, and the same 1p genotype was found in the majority of patients (12/18, 67%). The finding that primary carcinoma cells with metastatic ability usually contain 1p deletions, and that some cases lacking 1p alterations in the primary tumor acquire such changes during growth of a metastatic lesion, supports the notion that 1p loss may be important both early and late in colorectal carcinogenesis, with the apparent exception of local recurrences.

Adenocarcinoma↗

PubMedDate not supplied

Evaluation of breast cancer polyclonality by combined chromosome banding and comparative genomic hybridization analysis.

Cytogenetically unrelated clones have been detected by chromosome banding analysis in many breast carcinomas. Because these karyotypic studies were performed on short-term cultured samples, it may be argued that in vitro selection occurred or that small clones may have arisen during culturing. To address this issue, we analyzed 37 breast carcinomas by G-banding and comparative genomic hybridization (CGH), a fluorescent in situ hybridization--based screening technique that does not require culturing or tumor metaphases. All but two of the 37 karyotypically abnormal cases presented copy number changes by CGH. The picture of genomic alterations revealed by the two techniques overlapped only partly. Sometimes the CGH analysis revealed genomic imbalances that belonged to cell populations not picked up by the cytogenetic analysis and in other cases, especially when the karyotypes had many markers and chromosomes with additional material of unknown origin, CGH gave a more reliable overall picture of the copy number gains and losses. However, besides sometimes revealing cell populations with balanced chromosome aberrations or unbalanced changes that nevertheless remained undetected by CGH, G-banding analysis was essential to understand how the genomic imbalances arose in the many cases in which both techniques detected the same clonal abnormalities. Furthermore, because CGH pictures only imbalances present in a significant proportion of the test sample, the very detection by this technique of imbalances belonging to apparently small, cytogenetically unrelated clones of cells proves that these clones must have been present in vivo. This constitutes compelling evidence that the cytogenetic polyclonality observed after short-term culturing of breast carcinomas is not an artifact.

Breast Neoplasms↗

PubMedDate not supplied

Familial/bilateral and sporadic testicular germ cell tumors show frequent genetic changes at loci with suggestive linkage evidence.

Testicular germ cell tumor (TGCT) is the most common tumor type among adolescent and young adult males. Familial clustering and bilateral disease are suggestive of a genetic predisposition among a subgroup of these patients, but susceptibility genes for testicular cancer have not yet been identified. However, suggestive linkage between disease and genetic markers has been reported at loci on chromosome arms 3q, 5q, 12q, 18q, and Xq. We have analyzed primary familial/bilateral (n=20) and sporadic (n=27) TGCTs, including 28 seminomas and 19 nonseminomas, for allelic imbalance (AI) within the autosomal regions. DNA from all tumors were analyzed by fluorescent polymerase chain reaction of 22 polymorphic loci at 3q27-ter, 5q13-35.1, 12q21-ter, and 18q12--ter. All tumor genotypes were evaluated against their corresponding constitutional genotypes. The percentages of TGCTs with genetic changes at 3q, 5q, 12q, and 18q, were 79%, 36%, 53% and 43%, respectively. The frequencies at 3q and 12q in nonseminomas were significantly higher than in seminomas (P=.003 and P=.004). In order to evaluate changes at hemizygous Xq loci, five loci were analyzed by co-amplification with an autosomal reference marker known to reveal retained heterozygosity in the tumor DNA. Gain of Xq sequences was seen in more than 50% of the tumors. The degree of amplification varied among the loci in each of five tumors, and based on these breakpoints, a common region of overlapping gains was found at Xq28. No significant differences were found between the frequencies of genetic changes in familial/bilateral versus sporadic tumors, an observation speaking in disfavor of the existence of a single susceptibility gene for TGCT in any of the analyzed regions. Our data suggest that gain of genetic material at distal Xq and losses at 5q and 18q contribute to establishment of seminomas, whereas imbalances at 3q as well as gain at distal part of 12q are associated with further progression into nonseminomas.

Adult↗