Modification of gene expression induced by siRNA targeting of estrogen receptor alpha in MCF7 human breast cancer cells.

To establish a model of endocrine resistant breast cancer that is associated with loss of estrogen receptor (ER), MCF7 cells were transfected with several plasmid constructs intended to produce intracellular double stranded hairpin RNA to be processed into siRNA directed against different regions of the ERalpha mRNA. Stably transformed cells were propagated in long-term culture. One of these lines, designated pII, was selected for further analysis. pII cells exhibited reduced levels of ERalpha mRNA and protein as well as several estrogen-regulated genes assessed by real-time PCR and were unresponsive to addition of estradiol and tamoxifen. Higher levels of ERbeta were measurable as compared with parental MCF7 cells. There was an unexpected decrease in expression in members of the EGFR family in contrast with observations reported for ER-negative tumours or some other established endocrine-independent lines. Microarray gene analysis comparing expression in parental MCF7 with pII cells in both serum-synchronised and non-synchronised conditions highlighted a spectrum of other genes that were expressed at different levels compared to the parental MCF7 cells. Genes showing the greatest change were mostly common between synchronized and unsynchronised cells; GRB7, PSMD7, KRT19, KRT18, AKT1, SYNCRIP, CYB5A and EVL for down-regulated in pII and QDPR, VIM, CD68, CA9, STMN1, CDK2, CTSC for up-regulated in pII cells. Notably, the decreased expression of epithelial keratins 18 and 19 and an increase in vimentin and in a macrophage marker CD68, is suggestive of an epithelial to mesothelial transition. Further characterisation of these cells particularly with respect to the factors controlling their growth may contribute to a better understanding of the behaviour of cells that have become endocrine independent by loss of ER function.

Epidemiological and genetic study of 121 cases of oral clefts in Kuwait.

AIM: The aim of the study was to ascertain some epidemiological factors such as sex and consanguinity that may be associated with cleft lip with or without cleft palate (CL +/- CP) in Kuwait as well as to conduct genetic segregation analysis of these families. SETTING AND SAMPLE POPULATION: A total of 113 families ascertained through 121 CL +/- CP and CP surgical probands in Kuwait. The frequencies of cleft types and the epidemiological variables were calculated using SPSS version 5.0 software. Chi-square for goodness-of-fit test was used to test the significance of the associated epidemiological variables to facial clefts. Genetic segregation analysis was performed on 76 families with extended pedigrees and included only those with non-syndromic CL +/- CP (NS CL +/- CP). Major locus segregation analysis was used to fit models to the observed family patterns under Class A regressive models as implemented by REGD routine in S.A.G.E. release 4.0. A test for heterogeneity was also conducted to complete data set in addition to two subsets: Arabs and nomads. RESULTS: Of the 121 patients, 34(28.1%) had CP, 30(24.8%) had CL and 57 (47.1%) had CL + CP. The male to female ratio was 0.89 for CP, 1.14 for CL, 1.35 for CL + CP and 1.2 for all the clefts. The percentage of consanguineous families among those with a positive family history (60%) was not significantly different from that of the general population (54.3%), whereas for all the families with clefts the percent consanguineous was significantly lower (38%). No evidence of heterogeneity in the results between the Arab and nomad subsets was observed. The results for the major locus segregation analysis were inconclusive. CONCLUSION: No definite association was observed between consanguinity and the occurrence of facial clefts in Kuwait. General transmission models in the full data set showed no evidence of heterogeneity in the results between the Arab and nomad subsets.

Mutagenic effect of aflatoxin G1 in comparison with B1.

Available data indicate structure activity relationships in toxicity and carcinogenecity of aflatoxins in the order of B1 greater than G1 greater than G2 greater than B2. This set of tools is useful for investigations in experimental chemical mutagenesis. The present study is the second in a series concerning the activity of this group of aflatoxins on human chromosomes, using the techniques of Geimsa banding and sister chromatid exchanges. The results showed that aflatoxin G1 is a mutagenic agent to human chromosomes. The distribution of breaks on individual chromosomes according to relative length was nonrandom. Chromosomes 1, 2, 3, 4, and 5, the largest chromosomes, have more breaks, whereas chromosomes 19, 20, 21, and 22, the smallest chromosomes, have less than the expected number of breaks. Comparing these results with that of aflatoxin B1, we found that (1) the mutagenic activity of B1 on human chromosomes is higher than that of G1, and (2) although chromosomes 2, 11, 19, and 20 were the most affected by aflatoxin B1, chromosomes 1, 2, 3, 4, and 5 were the most affected by aflatoxin G1. This result indicates structure activity relationships in mutagenecity that are in agreement with those of the toxicity and carcinogenecity of aflatoxins B1 and G1.

Non-random distribution of cyclophosphamide-induced chromosome breaks.

The mutagenic effect of Endoxan on human lymphocytes was studied both in vivo and in vitro by the QM banding technique following the usual Giemsa stain. Chromatid breaks and interchanges were observed after applications in vivo. Chromosome aberrations were not distributed at random, because of a significant increase in the number of breaks on chromosome 15. The breakpoints affected the weakly fluorescent region on the chromosomes more than the strongly fluorescent region.

Mutagenic effect of aflatoxin B1.

The present work aimed to study the mutagenic effect of aflatoxin B1 on human chromosomes. The experiments showed that aflatoxin B1 is a strong chromosome damaging agent. The treated cells showed a high rate of aberrations mainly breaks and interchanges. Using Giemsa banding technique, the study showed that the distribution of breakage points on individual chromosomes was significantly non-random. The study of sister chromatid exchanges (SCEs) indicated that the high incidence of breakage rate was paralled by an increased SCE rate. Some chromosomes were very sensitive to the mutagenic effect of aflatoxin B1, while other chromosomes were very resistant. The present data provides an additional consideration in assessing the risks of exposure to this agent.