Jerry A. Styles

Gene expression and amplification in breast carcinoma cells with intrinsic and acquired doxorubicin resistance

The multidrug resistance (MDR) phenotype is a major cause of cancer treatment failure. Here the expressions of 4224 genes were analysed for association with intrinsic or acquired doxorubicin (DOX) resistance. A cluster of overexpressed genes related to DOX resistance was observed. Included in this cluster was ABCB1 the P-glycoprotein transporter protein gene and MMP1 (Matrix Metalloproteinase 1), indicative of the invasive nature of resistant cells, and the oxytocin receptor (OXTR), a potential new therapeutic target. Overexpression of genes associated with xenobiotic transformation, cell transformation, cell signalling and lymphocyte activation was also associated with DOX resistance as was estrogen receptor negativity. In all carcinoma cells, compared with HBL100 a putatively normal breast epithelial cell line, a cluster of overexpressed genes was identified which included several keratins, in particular keratins 8 and 18 which are regulated through the ras signalling pathway. Analysis of genomic amplifications and deletions revealed specific genetic alterations common to both intrinsic and acquired DOX resistance including ABCB1, PGY3 (ABCB4) and BAK. The findings shown here indicate new possibilities for the diagnosis of DOX resistance using gene expression, and potential novel therapeutic targets for pharmacological intervention.

Chemoprevention of breast cancer by tamoxifen : Risks and opportunities

ABSTRACT The antiestrogen tamoxifen is widely used in the adjuvant therapy of breast cancers in women and helps to prevent the occurrence of breast tumors in healthy women. However, epidemiological studies have shown tamoxifen treatment to be associated with a 2- to 5-fold increased risk of endometrial cancer. In rats but not in mice, long-term administration of tamoxifen results in an increase in hepatocellular carcinomas. Mechanistically, this occurs through metabolic activation of the drug, mainly by the CYP3A family, to an electrophilic species, that causes DNA damage in target tissues, and subsequently leads to gene mutations. It is controversial whether low levels of DNA damage occur in human uterine tissues, and there is no evidence that this can be causally related to the mechanisms of carcinogenesis. In healthy women, the risk:benefits for the use of tamoxifen is in part related to the risk of developing breast cancer. The results from the carcinogenicity studies in rats do not predict the likelihood that women will develop liver cancer or indeed cancers in other organs. The mechanism of endometrial cancer in women remains unresolved, but the experience with tamoxifen has highlighted the potential problems that need to be addressed in the assessment of future generations of selective estrogen receptor modulators.

Changes in transcription profile and cytoskeleton morphology in pelvic ligament fibroblasts in response to stretch: the effects of estradiol and levormeloxifene

Failure of ligamentous support of the genital tract to resist intra-abdominal pressure is a plausible underlying mechanism for the development of pelvic organ prolapse, but the nature of the molecular response of pelvic tissue support remains unknown. We hypothesized that the expression of genes coding for proteins involved in maintaining the cellular and extracellular integrity would be altered as a result of mechanical stretch. Therefore, cDNA microarrays were used to examine the difference in transcriptional profile in RNA of primary culture fibroblasts subjected to mechanical stretch and those that remained static. Out of 34 mechano-responsive genes identified (P < 0.05), four were coding for regulation of actin cytoskeleton remodelling, and its interaction with the extracellular matrix proteins; these are phosphatidyl inositol-4-phosphate 5-kinase (PIP5K1C), the human signal-induced proliferation associated gene-1 (SIPA-1), TNFRSF1A-associated via death domain (TRADD) and deoxyribonuclease 1-like 1 (DNase 1-L1). The transcriptosomal changes led us to investigate the phenotypic consequences of stretch, levormeloxifene and estradiol (E(2)) on the cytoskeleton of cultured fibroblasts. The percentage of cells with abnormal F-actin configuration was significantly higher in fibroblasts subjected to stretch compared with the static model (P < 0.0001). Levormeloxifene caused similar significant alterations in actin morphology of the static fibroblasts. The use of E(2) did not reverse the process or protect the cells from the effect of stretch, but significantly increased the rate of fibroblast proliferation, suggestive of a role in healing process. Mechanical stretch and/or levormeloxifene disturb the fibroblasts ability to maintain the cytoskeleton architecture and we speculate that they may disrupt ligamentous integrity and result in clinical prolapse.

Ishikawa cells exhibit differential gene expression profiles in response to oestradiol or 4-hydroxytamoxifen

In this study, the oestrogen agonist/antagonist action of 4-hydroxytamoxifen (OHT; 1 x 10(-6) M) and 17beta-oestradiol (E(2); 1 x 10(-8) M) were assessed on the oestrogen receptor (ER)-positive epithelial cell line (Ishikawa) with respect to cell proliferation, and to gene and protein expression. qRT-PCR and western blotting confirmed that Ishikawa cells expressed both ER isoforms and that there was no change in transcript levels in response to either ligand. Gene expression profiles, using oligonucleotide arrays representing approxiamtely 19,000 human genes, showed that the expression of 716 and 534 genes were changed differentially by treatment with either OHT or E(2) respectively, at the 24-h time point, with modulation of 46 genes common to both ligands, whereas 335 (OHT) and 240 (E(2)) genes showed expression changes unique to ligand, with 13 common alterations at 48 h. Both OHT and E(2) had demonstrable oestrogen agonist actions on Ishikawa cells, exemplified by increased proliferation and expression of known oestrogen-responsive genes, such as creatine kinase B and by the induction of alkaline phosphatase activity. Additionally, the data indicate that the two oestrogen agonists generated not only common gene expression changes but also unique ligand-specific profiles, raising the intriguing possibility that tamoxifen has E(2)-independent effects on the uterine epithelium.

Mutational specificity: Mutational spectra of tamoxifen-induced mutations in the livers oflacl transgenic rats

Tamoxifen, an important drug in breast cancer treatment, causes liver cancer in rats. The standard range of in vitro tests have failed to show that it causes DNA damage, but 32P‐postlabelling and DNA‐binding studies have shown that tamoxifen forms DNA adducts in rat liver. In 1995 a transgenic rat (Big Blue™; Stratagene, La Jolla, CA) became available which harbours the bacterial lacl gene, thereby allowing the in vivo study of tamoxifen mutagenesis. Recently, we [Styles JA et al. (1996): Toxicologist 30; 161] showed that tamoxifen caused an increase in the mutation frequency at the lacl gene in these transgenic rats. In this study, we report on our preliminary analysis of the mutational spectra of 33 control and 38 tamoxifen‐induced mutant lacl genes. Plasmid DNA containing the lacl gene was isolated from the mutant phages and its DNA sequence determined. In the control animal group, 81% of the mutant lacl genes were point mutations, whilst in the tamoxifen‐treated group, 62% of the mutant lacl genes were point mutations. Of the tamoxifen‐induced mutants, 43% were GC → TA transversions and 70% of point mutations. In the control group, GC → TA transversions were 19% of all mutations and 24% of point mutations. Thus, compared with control animals, tamoxifen treatment had significantly increased the proportion of GC → TA transversions.

Tamoxifen mutagenesis and carcinogenesis in livers of lambda/lacI transgenic rats: selective influence of phenobarbital promotion

Administration of tamoxifen (TAM) (20 mg/kg per day p.o.) for 6 weeks to female lambda/lacI transgenic rats caused a 4-fold increase in mutation frequency (MF) at the lacI gene locus in the livers of dosed animals compared with controls. After cessation of dosing, the MF showed a further increase with time at 2, 12 and 24 weeks, respectively. Phenobarbital promotion of similarly treated animals resulted in no increase in mutation frequency compared with TAM alone. Treatment with phenobarbital or TAM+phenobarbital resulted in time-dependent increases in liver weight compared with the corresponding controls. There was an increase in cell proliferation in the phenobarbital and TAM+phenobarbital groups, and at 24 weeks in the TAM dosed animals compared with controls. There was also a progressive increase in the number of GST-P expressing foci in the livers of TAM and TAM + phenobarbital rats compared with controls. The induction of cell proliferation and GSTP foci in the rat liver by phenobarbital is consistent with its ability to promote tamoxifen-initiated liver tumours in the rat. If the lacI gene is regarded as being representative of the rat genome in general (albeit that the gene is bacterial) the above observations suggest that promotion by tamoxifen confers selective advantage on mutated genes at loci that contribute to the tumour phenotype and that promotion of rat liver tumours by tamoxifen is not dependent simply upon the enhancement of cellular proliferation.

A microarray analysis of differential gene expression associated with the development of doxorubicin resistance in breast carcinoma

One of the major complications of cancer chemotherapy is the development of a multidrug resistance (MDR) phenotype. Genes known to be major contributors to this phenotype include the ABC transporter family, DNA repair class genes and phase II conjugators such as the GST family. However, though there may be predominant contribution to the MDR phenotype from one gene or family the overall phenotype probably results from the contribution of a cohort of genes, both of downstream effecter and upstream regulatory classes. Additionally, other genes differentially expressed in the MDR verses drug sensitive states may result in other phenotypic traits that may also complicate treatment, for example an increased propensity towards invasiveness. We have applied microarray analysis to breast carcinoma cells with intrinsic and acquired doxorubicin resistant phenotypes and compared the gene expression profiles obtained with each other, and with those gathered from various other drug sensitive breast carcinoma cell lines (Turton et al. 2001). The analysis has been applied at both the mRNA and genomic levels. Some expected changes in gene expression (mRNA analysis) and amplification (genomic DNA analysis) such as that for the multidrug resistance 1 (ABCB1) gene were detected. However, some unexpected associations of differential gene expression were found, some associated with known phenotypic traits, and some that may offer alternative targets for chemotherapy. The similarity of gene expression profiles between the cells with intrinsic and acquired doxorubicin resistance indicated that a tumor with an acquired doxorubicin resistant phenotype arises as a result of drug driven selection of cells with an intrinsic resistant phenotype from the majority drug sensitive population.

Strategy for nuclear proliferation

Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events have not been completed. The tumour suppressor p53 has been implicated in a Gl checkpoint. To investigate whether P53 also participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors. The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.