Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1.

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells.

Feasibility of simultaneous fluorescence immunophenotyping and fluorescence in situ hybridization study for the detection of estrogen receptor expression and deletions of the estrogen receptor gene in breast carcinoma cell lines.

For the first time, combined immunophenotyping and fluorescence in situ hybridization (FISH) technique according to the "fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms" (FICTION) technique have been successfully applied in solid tumors. Thus, we were able to visualize the antigen expression of cells with chromosomal deletions of a tumor suppressor region directly. In six breast carcinoma cell lines, we investigated the correlation between estrogen receptor (ER) expression status and deletions of the estrogen receptor gene (ESR). To screen for deletions of the ESR gene, dual-color FISH was performed with a YAC (yeast artificial chromosome) probe containing the ESR gene and, as internal control, with a centromeric probe of chromosome 6. Deletions of the ESR gene were detected in four of six cell lines. For direct comparison of ER expression with the copy number of the ESR gene at the single cell level, immunophenotyping with mouse anti-human ER antibody was combined with FISH with the YAC probe containing the ESR gene according to the FICTION technique. There was no correlation between lack of or reduced ER expression and deletions of the ESR gene. One cell line with deletions of the ESR gene did express ER on the protein level, while another cell line without a deletion did not. Cells with deletions of the ESR gene were either ER expression positive or negative. The staining intensity of ER expression was not associated with the copy number of the ESR gene. Thus, this FICTION study unequivocally shows that deletions of the ESR gene are not the major cause of absent or reduced ER expression in breast carcinoma cell lines.

Detection of 6q deletions in breast carcinoma cell lines by fluorescence in situ hybridization.

Dual-color fluorescence in situ hybridization was performed to detect the frequency and extent of 6q deletions in ten breast carcinoma cell lines. In five cell lines, the 6q deletions involved large regions extending from 6q12-q16 to 6q27, and in one the deletion extended from the region distal to YAC 751G10 at 6q25.1 to 6q27. In two cell lines, 6q deletions occurred only in cells with polysomy 6, indicating that such deletions might be secondary chromosomal aberrations and reflect late genetic changes in breast carcinomas. In addition, an overrepresentation of 6q21-q22.2 was detected in one cell line.

Hypertension in Alaska Natives: association with overweight, glucose intolerance, diet and mechanized activity.

OBJECTIVE: Determine the prevalence of hypertension in Alaska Natives and evaluate risk factors. DESIGN: Population-based univariate and multivariate analysis of blood pressure in 1124 Alaska Natives over 20 years of age. RESULTS: The sample had mean: age 45 years, body mass index 27, systolic pressure 123 mmHg and diastolic pressure 73 mmHg. The age-adjusted rate of hypertension > or = 160/95 mmHg was 9.1% and 6.8% among Athabascan Indians and Yup'ik Eskimos, respectively. After controlling for age and sex there was significantly more hypertension among Athabascan Indians (OR = 1.53, CI = 1.07-2.2, p = 0.019) compared to Yup'ik Eskimos. Race was significantly associated with blood pressure > or = 140/90 when controlled for age and overweight (p = 0.01, OR = 0.78, CI = 0.69-0.95). The presence of hypertension was significantly associated with the following: intake of non-indigenous food (p = 0.01); mechanized activities (p = 0.01); and glucose intolerance in both women (p = 0.043) and men (p = 0.001). Multiple regression analysis revealed age (OR = 1.06, CI = 1.05-1.08) and overweight in both men (OR = 3.02, CI = 1.85-4.93) and women (OR = 2.76, CI = 1.81-4.19) to be significantly associated with BP > or = 140/90. CONCLUSION: Hypertension is no longer rare in Alaska Natives and is associated with overweight, non-indigenous diet, mechanized activities, and glucose intolerance.

A defined chromosome 6q fragment (at D6S310) harbors a putative tumor suppressor gene for breast cancer.

Recent evidence obtained by cytogenetic and molecular studies indicates that in breast cancer chromosome 6q is often affected by genetic changes suggesting the existence of putative tumor suppressor genes (TSGs). However the function of gene(s) on this chromosome in breast cancer suppression is not understood. To substantiate further the presence of breast cancer related TSGs at 6q and to define their location, we first performed microcell-mediated transfer of chromosome 6 to CAL51 breast cancer cells for studying possible suppression of malignant phenotype and secondly, we analysed DNAs from 46 primary breast cancers for loss of constitutive heterozygosity (LOH) using 24 poly-morphic microsatellite markers. The chromosome transfer resulted in loss of tumorigenicity and reversion of other neoplastic properties of the microcell hybrids. Polymorphism analysis of single hybrids revealed that they harbored only a small donor chromosome fragment defined by the marker D6S310 (6q23.3-q25) and flanked by D6S292 and D6S311. The LOH data suggest that four tumor suppressor gene loci mapped to the central and distal portion of 6q may be independently deleted in breast cancer. One of these regions corresponds to the region identified by chromosome transfer.

Suppression of tumorigenicity of breast cancer cells by transfer of human chromosome 17 does not require transferred BRCA1 and p53 genes.

A number of candidate tumor suppressor genes located on the human chromosome 17 are thought to have a role to play in the development of breast cancer. In addition to the p53 gene on 17p13.1 and the BRCA1 gene mapped to 17q12-21, other chromosomal regions for tumor suppressor genes have been suggested to exist on 17p13.3 and both the central and the distal parts of 17q, although definitive functional proof of their involvement in breast cancer tumorigenesis is still lacking. In this report we show that microcell transfer of a human chromosome 17 into wild-type p53 breast cancer cells CAL51 results in loss of tumorigenicity and anchorage-independent growth, changes in cell morphology and a reduction of cell growth rates of the neo-selected microcell hybrids. In the hybrid cells, which express the p53 wild-type protein, only the p- and the distal parts of the q arm of donor chromosome 17 are transferred. Thus, our results provide functional evidence for the presence of one or more tumor suppressor gene(s) on chromosome 17, which are distinct from the p53 and the BRCA1 genes.

Wild-type p53 is not involved in reversion of the tumorigenic phenotype of breast-cancer cells after transfer of normal chromosome-17.

A number of different candidate tumor suppressor genes involved in human breast cancer are presumed to be located on chromosome 17. To verify the relevance of chromosome 17 abnormalities in breast cancer cells, a normal human chromosome 17 was transferred by microcell fusion to R30 tumor cells derived from an infiltrating ductal mammary carcinoma. The tumorigenicity of the microcell hybrids in nude mice was examined. The tumor volume obtained with different clones was reduced by up to 94% of the value corresponding to the parental tumor cells. This effect was accompanied by a reduction of anchorage-independent growth, as well as cell growth rates on plastic plates. These effects were independent of the continued presence of a transferred 17q arm and could not be attributed to the action of the normal p53 gene. The results support the assumption that in addition to p53 a further tumor suppressor gene is located on 17p which is involved in breast cancer.

Mutagenic activity of BKV and JCV in human and other mammalian cells.

We present data suggesting that human polyomaviruses BKV and JCV, widely distributed throughout human populations, are able to induce gene mutations in cultured cells. In this study, using different infecting agents, cell lines to be infected, mutation expression periods, and selection systems, we observed mutagenic effects of varying extent with values of spontaneous mutant frequencies being increased after BKV infection up to 100-fold in BHK cells (6-thioguanine resistance) and nearly 35-fold in virus-transformed human Lesch-Nyhan cells (ouabain resistance). In experiments with BKV the viral mutagenic potential was found to be raised both in moderately uv-irradiated cells, or when wild-type virus was replaced by the variant BKV-IR isolated from a human tumor. Since BKV-IR is defective in the expression of small-t antigen, the viral mutagenicity does not require this protein to be active. BKV was shown to mutate, besides different established cell lines, human peripheral blood lymphocytes. Moreover, as demonstrated by comparing mutagenicities of DNAs from BKV, JCV, and the related polyomavirus SV40, the mutagenic effects of the three viruses do not appear to be essentially different. Implications of these findings are discussed.

Simian virus 40-induced mutagenesis: action of the early viral region.

Earlier results have demonstrated a mutagenic activity of simian virus 40 (SV40) in mammalian cells. To analyse this ability further, the effect of SV40 DNA fragments, introduced into Chinese hamster cells, on the frequency of mutations at the hypoxanthine phosphoribosyltransferase locus and other loci was studied. It was found that the mutagenic effect was substantially maintained when the viral genome had been replaced by a fragment comprising the T antigen-coding region and the early promoter-enhancer region; was strongly reduced or abolished when the promoter region including upstream sequences in this fragment had been replaced by the chicken lysozyme gene promoter or both enhancer elements were deleted, and was abolished in an SV40 replication origin-defective mutant in which the structure of the T antigen-binding site II was affected. It may be concluded that SV40-induced mutagenesis depends on the expression of the early region of the genome and on a function involved in specific binding of large T antigen to viral DNA. Since origin-defective mutants of SV40 were reported as being able to transform cells, the functions of transformation and mutation do not seem to correlate.

In vitro transformation and mutation of Chinese hamster cells by different SV40 nucleoprotein complexes.

SV40 minichromosomes (MCH) either isolated from SV40 infected CV-I monkey cells (native MCH) or reconstituted in vitro from viral DNA and the H1 depleted calf thymus histone fraction could transform and mutate Chinese hamster (CH) cells in vitro. Whereas reconstituted MCH transformed and mutated CH cells with about the same efficiency as purified SV40 DNA, approximately 10-200-fold increase in the transforming activity had been demonstrated for native MCH. All transformed cell colonies and a major part of the isolated mutant cell clones recovered after inoculation of CH cells with SV40 MHC expressed the SV40 T antigen. Addition of H1 to both purified SV40 DNA and reconstituted MHC drastically diminished the transforming capacities of both agents. Possible reason(s) for the inhibition effect of H1 histone is discussed.

Are transforming and mutagenic activities of oncogenic papovaviruses correlated?

A number of results obtained recently have shown that SV40-induced mutation and transformation of mammalian cells cultivated in vitro are related in some respect. Experiments were undertaken in order to get further information on the mode of mutagenic action of papovaviruses and further evidence of correlations existing between the mutagenic and transforming viral activities. DNA from the oncogenic hamster papovavirus HaPV was found to be mutagenic for at least three different resistance markers. In the hamster cell lines which were used HaPV DNA produced a higher yield of mutants than SV40 DNA for both the azaguanine and the aminopterin resistance marker. Cellular clones carrying a SV40-induced mutation in a specific locus were found to harbour viral genetic material and to exhibit a genetic instability of other loci. Also, cell lines characterized by their SV40-transformed state did exhibit such genetic instability. These results and results of other authors are discussed with respect to the correlations between oncogenic virus-induced mutation and transformation.

Virus-induced gene mutations of eukaryotic cells.

Most animal viruses studied so far induce chromosomal aberrations. In addition, adenoviruses, papovaviruses, and retroviruses are known to induce gene mutations like mutagenic bacteriophages. At least in one case studied retrovirus induced mutagenesis involves gene and/or scripton splitting analogous to the mutagenic mechanism of action of mutatorphage Mu and other movable DNA elements. On the contrary, several results obtained by independent means indicate that Simian virus 40, a papovavirus, does not act by splitting the affected gene but presumably by generation of base pair substitutions or of other minor DNA damages leading to amino acid substitutions. The mechanisms involved are still unknown. There a some hints, however, that these mechanisms might have some step(s) in common with processes leading to malignancy. In fact those viruses proved unequivocally so far to be capable of inducing gene mutations are oncogenic viruses.

Influence of superinfection on the photoreversible phase of UV induced lysogenic bacteria.

1. Lysogenic induction by UV light can be reversed by photoreactivation. UV-treated E. coli K12 (lambda)+ uvr+ and uvr cells are sensitive to photoreactivation for a given time after irradiation. This sensitivity suddenly disappears at the end of this time. 2. The photoreversible period of UV induction is more than twice as long in uvr cells as it is in uvr+ cells. 3. The photoreversible period can be reduced by superinfection with lambda c mutants after irradiation. This effect is positively correlated with the multiplicity of superinfection. Such a reduction does not occur when superinfection is carried out with wild-type phages or with heteroimmune derivatives. 4. We concluded that during the photoreversible period of UV induction oligonucleotides are excised or synthesized and gaps are formed during excision repair and post replication repair of UV damage; these might react with E. coli recA protein thereby activating it to induce its own synthesis, to cleave phage repressors and to exert its other SOS functions.

DNA of simian virus 40 mutates Chinese hamster cells.

Infection of Chinese hamster cells with SV 40 DNA gives rise to mutants resistant both to S-axaguanine (AG) and aminopterin (AP). This mutagenic effect can be raised when facilitating DNA uptake of cells by a helper agent. The extent of muyagenic action depends further on the concentration of DNA applied to the cells, with 2 micrograms/ml being more effective than 10 micrograms/ml, as well as on the period of incubation of infected cells before onset of mutant selection (mutation expression time). Using the AG resistance marker the mutation frequency can be increased more than 8-fold compared with the spontaneous mutation frequency. Reconstituted SV 40 minichromosomes show a mutagenic action which is similar to the DNA-mediated mutagensis whereas non-viral DNA from mammalian cells fails to induce mutations significantly. A major part of isolated clones of SV 40-induced mutants tested so far does express SV 40 T-antigen, suggesting the persistence of SV 40 genetic material in these clones. The possible existence of relations between mutagenic and transforming capacities of SV 40 is discussed.

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoriboxyltransferase into mutant cells.

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly (L-lysine), lysolecithin and amphotericin B seem to inhibit the entry of the enzymes of their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10--20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.

Mutagenesis by simian virus 40. II. Changes in substrate affinities in mutant hypoxanthine-guanine phosphoribosyl transferase enzymes at different pH values.

A number of 8-azaguanine-resistant clones selected from Chinese hamster cells infected with SV 40, and supposed to originate by virus infection was investigated to demonstrate and analyze genetic alterations occurring in the cells after infection. All resistant clones tested showed reduced but detectable activity levels of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The extent of reduction in the activity was not identical for different substrates. In all the clones tested, spontaneous mutants included, the pH optimum for the enzymic reaction with guanine was shifted to lower values. The reduced enzymic activities of resistant clones correlated with their colony-forming ability in corresponding selective media. The results support the suggestion that SV 40 is able to induce gene mutations.

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.