Biomedical subjects
Publications and source records attributed to S Ingvarsson.
Using Southern blot analysis of DNAs from rat X mouse somatic cell hybrids, we have mapped Nmyc and Lmyc, two members of the myc family of proto-oncogenes, to rat chromosomes 6 and 5, respectively.
Postmitochondrial and cytosolic polypeptides were extracted from human breast tumors and non-malignant breast tissue and analyzed using high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Approximately 800-1000 postmitochondrial and 600-800 silver stained cytosolic polypeptides were detected over the pH range of 4.8 to 7.5 and molecular weight range of 18-120 kDa. The 2D-PAGE patterns of polypeptides from normal and malignant tissue were very similar, although both qualitative and quantitative polypeptide differences were noted. Six cytosolic polypeptides (pI/molecular weight X 10(-3) 5.20/80 kDa, 5.75/43, 6.25/40, 5.43/35, 5.45/34.5, 5.50/34 and 6.15/24 were expressed only in malignant tissues. One constitutive polypeptide, 7.25/52, was not detected in any of the malignant tissue samples. Quantitatively, marked differences in spot density were noted in polypeptides localized mainly in the molecular weight ranges of 22-40 kDa and pI ranges of 5.65-7.00. A general increase in polypeptide expression was noted in malignant tissues as compared to normal. Twenty-two polypeptides were significantly and consistently increased in tumor samples while only one polypeptide was decreased. One polypeptide, p24 (6.15/24) was expressed in greatest concentrations in tumors which also expressed the greatest estrogen receptor content. Expression of p24 was markedly reduced in normal tissue and malignant tissues expressing low levels of estrogen and progesterone receptors.
c-myc and N-myc are closely related genes coding for putative DNA-binding proteins. The protein products of both genes have been implicated in the regulation of growth of normal and neoplastic cells. We compared the regulation of N-myc and c-myc expression under different growth conditions as well as in vitro differentiation of the murine EC lines F9 and PCC7. N-myc and c-myc expression was found to be regulated by distinct mechanisms, although similarities exist. Differences were found both at the transcriptional and at the post-transcriptional level. The two myc genes were regulated by mainly post-transcriptional mechanisms, but in PCC7 cells nuclear run-on assays indicated that c-myc was repressed at the level of transcription. N-myc and c-myc expression was negatively regulated at a post-transcriptional level in F9 and PCC7 cells during differentiation to visceral endoderm and nerve-like tissue, respectively. Serum stimulation of F9 cells for 4 h induced a sevenfold increase in c-myc transcripts but no significant elevation of N-myc transcripts. Mitogenic stimulation with insulin and transferrin also induced a marked elevation of c-myc but not of N-myc mRNA. In addition, the N-myc and c-myc genes differed in F9 cells with respect to (i) the kinetics of expression following induction of differentiation, c-myc undergoing quicker changes than N-myc; (ii) the response to cycloheximide inhibition of protein synthesis, indicating that c-myc but not N-myc is down-regulated by a short-lived protein; and (iii) the half-lives of the transcripts, estimated to be approximately 40 min for c-myc and 130 min for N-myc.
Mouse Pvt-1 and rat MIS1 are frequent proviral integration sites in retrovirally induced lymphomas. The Pvt-1 locus is also involved in mouse plasmacytoma (6;15) and in the variant Burkitt lymphoma (2;8) translocations. We show that the Pvt-1/MIS1 locus is syntenic with MYC on rat chromosome 7. This is consistent with a postulate of close linkage and, possibly, a functional relationship between the MYC protooncogene and the MIS1/Pvt-1 locus.
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Spontaneously arising immunocytomas in Lou/Wsl rats contain a consistent translocation between chromosomes 6 and 7. The c-myc gene has been localized to chromosome 7 and has been shown to be rearranged in the majority of the rat immunocytomas. We now report the cloning of the rearranged 11-kilobase EcoRI c-myc fragment from the IgE-secreting IR75 tumor. Sequence analysis revealed that the cytogenetically visible t(6;7) translocation must have involved several events in this tumor. One event has led to the juxtaposition of c-myc and the switch mu region, in a head-to-head orientation. The breakpoint is approximately 850 base pairs upstream from the proximal c-myc promoter on chromosome 7. This area is distinct from the more common mouse plasmacytoma- and Burkitt lymphoma-associated translocation breakpoints and also differs from the known murine retroviral insertion sites. A second rearrangement has led to the transposition of sequences upstream from the switch gamma 1 region to the c-myc-distant end of the switch mu region, tail-to-tail. This requires at least two events, including one inversion. In addition to showing that identical loci (c-myc, immunoglobulin) are juxtaposed via chromosomal translocations in three different tumors (Burkitt lymphoma, mouse plasmacytoma, and rat immunocytoma) in different species (human, mouse, and rat), the multiple rearrangements in IR75 and some other tumors emphasize the selective value of c-myc activation by an immunoglobulin locus in the tumorigenic process.
The effect of the glucose analogue 5-thio-D-glucose (5TG) on the yeast Saccharomyces cerevisiae was studied. Derepression of mitochondrial respiratory chain cytochromes, alcohol dehydrogenase (isoenzyme II), NADH dehydrogenase and maltase was inhibited by 0.5-2 mM-5TG. Growth rate was only slightly affected. Ethanol was efficiently produced with 2 mM-5TG in medium initially containing 0.25% glucose. Mutants resistant to the growth inhibitory effects of 5TG on glycerol medium showed resistance to the catabolite repressing effects of glucose. Other mutants, known to be catabolite repression resistant, showed resistance to 5TG. The analogue seems to inhibit derepression of glucose repressible enzymes with greater potency than glucose itself.
Experiments have been carried out to establish the relative position of the mouse Ig heavy chain locus and the c-myc oncogene. In mouse plasmacytoma with the typical rcpt(12;15) chromosome translocation the c-myc oncogene is juxtaposed to one of the heavy chain genes in a head-to-head orientation. Since the relative orientations of the c-myc locus and the Ig heavy chain gene cluster on the corresponding mouse chromosomes had not been settled, it was not known whether the rearranged c-myc gene is transposed to chromosome 12 or remains on chromosome 15. To decide which of the two alternatives is correct, we separated the translocation chromosomes by fluorescence-activated chromosome sorting. The separated chromosomal fractions were hybridized with myc-specific DNA probes corresponding to the first or second/third exons in a chromosome spot assay. The results presented here indicate that the c-myc gene in mouse plasmacytoma carrying the typical translocation, as in the human Burkitt lymphoma analogous translocation, transposes to the chromosome carrying the Ig heavy chain locus. These results also establish the orientations of the Ig heavy chain locus and the c-myc locus on their normal chromosomes.
Subcellular distribution of hexokinase (HK) isoenzymes in 22 human breast cancers (21 primary cancers and 1 axillary metastatic growth) and 7 non-pathological human mammary gland tissue samples was studied with starch gel electrophoresis on isolated cell fractions obtained by differential centrifugation. Fractions used were cytosol, mitochondria and microsomes. A comparison of two methods for detecting HK activity was made, yielding different results regarding HK II and HK III. A method based on the ability of NADPH to fluoresce in UV gave a constant pattern of HK isoenzymes. In non-pathological breast tissue, only HK I was seen, i.e. in the cytosol and the microsomal fractions. HK I was also seen in all fractions of the cancers, but another more anodal band of HK I, as well as HK II and HK III, consistently appeared in the cytosol fractions. The more commonly used staining technique with tetrazolium dye revealed HK II in 45% and HK III in 50% of the samples. The pattern of HK isoenzymes in the cancers was the same irrespective of estrogen and progesterone cytosolic receptor contents and the histology of the tumors. The fluorescence method is, therefore, much more sensitive than the tetrazolium technique for detecting HK activity after electrophoresis and could explain difference in results obtained by various laboratories.
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Instability of microsatellite DNA or replication error (RER) is characteristic of tumours caused by mismatch repair (MMR) deficiency. Germline mutations in MMR genes are associated with Hereditary non-polyposis colorectal carcinoma (HNPCC) and somatic mutations in these genes are also found in a substantial fraction of colorectal cancers (CRC). In this study we concurrently screened colorectal tumours for the RER phenotype and loss of heterozygosity (LOH) at MMR gene loci. The RER phenotype was evident in 47/197 (24%) tumours. RER was more commonly detected in young patients (< 50 years) and in tumours located in the proximal colon. RER was positively associated with LOH at the hMSH2/hMSH6 loci on chromosome 2p, where LOH was observed in 46% of the RER+ tumours. LOH at hMLH1 and hPMS1 loci was more frequent in the younger patients (< 50 years). RER was not associated with clinicopathological parameters, such as Duke's stage and tumour differentiation (grade). The RER phenotype was associated with better overall survival, but there was a trend towards significance when multivariate analysis was used. This indicates that loss of MMR genes generate a less aggressive phenotype, and raises the question about RER being a useful indicator of prognosis for CRC patients.
Germline alterations of the BRCA1 or BRCA2 genes result in susceptibility to breast and ovarian cancer. Protein-protein interaction studies, transcription activity and mouse knockout experiments have suggested that the Brca1 and Brca2 proteins are of importance in DNA repair and maintenance of genome integrity, possibly due to the transactivation function of Brca1 or Brca2. Subsequently, tumors in individuals carrying germline mutation in either BRCA1 or BRCA2 gene show instability at chromosomal and gene level. Chromosomal and gene alterations are more pronounced in tumors from BRCA1 and BRCA2 mutation carriers than in sporadic tumors. Furthermore, BRCA1 and BRCA2 mutated breast tumors differ from sporadic tumors in respect to histological phenotype. Typically, a higher grade of malignancy is observed in familial tumors. This review summarizes the putative functions of the Brca1 and Brca2 proteins and pathogenesis in tumors of BRCA1 and BRCA2 mutation carriers.
In this study 238 human primary breast cancers were analysed with 9 polymorphic microsatellite markers specific to region 7q21-q35 on chromosome 7. LOH was observed at one or more marker in 82 cases or (34%). The deletions were evenly distributed throughout the region. Patients were divided into two groups according to whether LOH was observed in their tumours or not, and tested for association with overall survival, the clinicopathological features: steroid receptor content, tumour size, node status, DNA ploidy and S-phase fraction, and LOH at other chromosomal regions. An association was found between 7q LOH and high S-phase fraction. An association was found between LOH at 7q and LOH at 1p, 3p, 9p, 13q and 17q. These results suggest the location of a putative tumour suppressor gene at chromosome 7q21-q35 that, in combination with other deletions, might enhance tumour growth.
Human primary breast cancers were analysed for somatic loss of heterozygosity (LOH) at chromosome 18 with 15 polymorphic microsatellite markers. LOH was observed in 148 of the 228 cases analyzed, (65%). Three smallest common deletion regions (SCDR) were detected on the long arm of chromosome 18. The marker D18S51 at the region 18q22 showed the highest LOH (42%). Tumors with and without LOH at 18q were tested for association with clinico-pathological features of the tumors, such as estrogen and progesterone receptor content, age at diagnosis, tumor size, node status, histological type, S-phase fraction, DNA ploidy and LOH at other chromosomal regions. A significant association was found between LOH at 18q and high S-phase fraction (99.9% confidence interval) and low progesterone receptor content (99% confidence interval). Furthermore, an association was found between LOH at 18q and LOH at 1p, 7q, 9p, 13q and 17q. We conclude that there are three separate LOH target regions at chromosome 18q, and that inactivation of one or more genes at these regions might be important for human breast carcinogenesis.
Replication errors (RER) at microsatellite repeats indicate genomic instability in hereditary nonpolyposis colorectal cancer (HNPCC) and in some sporadic cancers. We have studied genomic instability in 313 sporadic breast tumors and in 106 tumors from BRCA2, 999del5 carriers at 43 genomic loci on 13 chromosomes. RER was observed in 8/419 (1.9%) of the cases at one or more chromosomal loci. The frequencies of type I and type II RER were similar. The majority of RER+ tumors showed ER+, PgR+, high S-phase fraction, tumor size >2 cm and LOH at 2p, 2q and 3p. All 8 RER+ tumors were of the ductal histotype. The breast cancer cases with RER are not part of an HNPCC syndrome and a family history of colorectal cancer growth is not detected in relatives, with the exception of one case. However, four of the RER+ cases are from individuals carrying the BRCA2, 999del5 mutation. We conclude that RER is a rare somatic event during human breast carcinogenesis and may be associated with progression of breast carcinomas.