Reginald Davies

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.

Essential Role of the AH Receptor in the Dysfunction of Heme Metabolism Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The dysfunction of hepatic heme synthesis by 2,3,7,8-tetrachlordibenzo- p-dioxin (TCDD) in mice, enhanced by iron, leads to accumulation of uroporphyrins I and III (uroporphyria) and resembles the human disorder porphyria cutanea tarda (PCT) precipitated by alcohol and estrogenic drugs. Although consequences of TCDD are considered entirely dependent on the aryl hydrocarbon receptor (AHR), this is not proven for uroporphyria. Administration of TCDD (75 microg/kg) caused uroporphyria in susceptible C57BL/6J mice with high-affinity AHR after 5 weeks (>600-fold increase in hepatic uroporphyrins). Transcriptomics showed significant modified gene expressions for intermediary, heme, and iron metabolism as well as for oxidative stress and cell injury. Resistant low-affinity AHR DBA/2 mice (no increase in porphyrins) showed far fewer changes. At this dose of TCDD, persistent up-regulation of some traditional AH battery genes occurred in both strains. Essentiality of AHR was demonstrated with C57BL/6 Ahr knockout mice. Elevation of hepatic uroporphyrins was 964-fold in Ahr (+/+) mice, lower in Ahr (+/-) (60-fold), but undetectable with Ahr (-/-) . Consistent with an oxidative mechanism, iron overload enhanced porphyria as well as general liver injury in Ahr (+/+) and Ahr (+/-) mice but had no interactive effect in Ahr (-/-) . In contrast, when iron-treated mice received, instead of TCDD, the heme precursor 5-aminolevulinic acid (ALA), causing uroporphyia in Ahr (+/+) mice (242-fold rise in uroporphyrins), elevation of uroporphyrins I and III (42-fold) also occurred in Ahr (-/-) mice and was seemingly associated with AHR-independent expression of Cyp1a2. The findings prove that AHR is a key factor in porphyria induced in mice by TCDD. However, in other models of human PCT, participation of AHR may not be an essential requirement.

LIGHT PULSE-INDUCED HEME AND IRON-ASSOCIATED TRANSCRIPTS IN MOUSE BRAIN: A MICROARRAY ANALYSIS

Synchronization of circadian oscillators with the outside world is achieved by the acute effects of light on the levels of one or more clock components. In mammals the PAS transcription factors Clock, NPAS2, and BMAL1 regulate gene expression as a function of the day‐night cycle. Both PAS domains of NPAS2 were found to bind heme as a prosthetic group, form a gas‐regulated sensor, and exert heme‐status control of DNA binding in vitro. In a microarray analysis comparing overall changes in brain transcript levels between mice subjected to light pulses during the dark phase with animals maintained in darkness, we traced consistent changes in more than 200 different transcripts. Of these, 20 are associated with heme and iron biosynthesis and catabolism. A model for the pathway of induction of heme and iron homeostasis‐related transcripts resulting from light pulses suggests that light signals (as stressors) induce transcription of heme oxygenase 2 (Hmox2) and cytochrome P450 oxidoreductase (Por), which may serve as a primary line of cellular defense. HMOX2 degrades heme from proteins such as hemoglobin. This degradation generates CO, a signal molecule, and may also change the redox state of the cell by reducing the NADPH/NADP ratio. This could lead to up‐regulation of globin gene transcription, thereby releasing iron that in turn controls production of ferritins, and further up‐regulating aminolevulinate synthase 2 (Alas2).

Multiple polymorphic loci determine basal hepatic and splenic iron status in mice

Polymorphisms of genes linked to iron metabolism may account for individual variability in hemochromatosis and iron status connected with liver and cardiovascular diseases, cancers, toxicity, and infection. Mouse strains exhibit marked differences in levels of non‐heme iron, with C57BL/6J and SWR showing low and high levels, respectively. The genetic basis for this variability was examined using quantitative trait loci (QTL) analysis together with expression profiling and chromosomal positions of known iron‐related genes. Non‐heme iron levels in liver and spleen of C57BL/6J × SWR F2 mice were poorly correlated, indicating independent regulation. Highly significant (P < .01) polymorphic loci were found on chromosomes 2 and 16 for liver and on chromosomes 8 and 9 for spleen. With sex as a covariate, additional significant or suggestive (P < 0.1) QTL were detected on chromosomes 7, 8, 11, and 19 for liver and on chromosome 2 for spleen. A gene array showed no clear association between most loci and differential iron‐related gene expression. The gene for transferrin and a transferrin‐like gene map close to the QTL on chromosome 9. Transferrin saturation was significantly lower in C57BL/6J mice than in SWR mice, but there was no significant difference in the serum level of transferrin, hepatic expression, or functional change in cDNA sequence. β2‐Microglobulin, which, unlike other loci, was associated with C57BL/6J alleles, is a candidate for the chromosome 2 QTL for higher iron. In conclusion, the findings show the location of polymorphic genes that determine basal iron status in wild‐type mice. Human equivalents may be pertinent in predisposition to hepatic and other disorders. (HEPATOLOGY 2006;44:174–185.)

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.

The preparation of highly enriched fractions of binucleated rat hepatocytes by centrifugal elutriation and flow cytometry.

A procedure is described for the isolation of highly enriched fractions of binucleated hepatocytes from rat liver. Liver cells isolated by EGTA and collagenase perfusion were initially subjected to centrifugal elutriation and second to flow cytometry coupled with Hoechst 33342 staining. The elutriation step yielded hepatocyte fractions which contained almost entirely mononuclear diploid cells and fractions enriched in binucleate hepatocytes. The fractions with the highest proportion of binucleated hepatocytes contained between 50 and 56% of these cells. Subsequent flow cytometric cell sorting yielded fractions which contained greater than 80% binucleated cells. These cells were viable in culture as demonstrated by the immunohistochemical detection of bromodeoxyuridine incorporation.

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.

Use of reverse genetics and cDNA arrays to understand ‘dioxin’ toxicity

The molecular mechanisms of toxic agents are rarely likely to be the result of change in the expression of a single gene. Even apparently simple actions of chemicals are probably the consequence of genetically variable multigene expression. One strategy for attacking this problem is to take advantage of the genetic variation of response in mice to search for susceptibility genes using genetic linkage analysis in combination with comparisons of gene expression in the parent strains by cDNA microarray technology. Genetic variation in mice and toxicogenomics were used to explore mechanisms of gene interaction leading to cell malfunction and injury in the liver caused by dioxin. This demonstrated susceptibility loci, other than the Ahr gene, pertinent to the development of porphyria (a disruption of heme synthesis) and liver injury. cDNA arrays of 4000 IMAGE clones pertinent to toxicology were used to compare candidate multiple gene expression in strains relative to their initial hepatic response e.g. induction of drug metabolism enzymes, and to their subsequent development of porphyria and liver injury. Phenotypic response was compared with gene expression by metabolic system including groups of genes for heme and iron metabolism, the AH battery and oxidative stress.

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.

Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGFβ1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGFβ1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGFβ1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGFβ1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFGβ1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B1 retained their sensitivity to the effects of TGFβ1. Thus the loss of the inhibitory effect of TGFβ1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.