Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 microM, resulted in stably resistant cell lines (MCFTDX and H630TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCFTDX and a 52-fold increase in TS levels in H630TDX cell lines. Despite continued exposure of these cells to 2.0 microM TDX, TS protein and TS mRNA expression decreased to parental levels in H630TDX cells, whereas in MCFTDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCFTDX cell line. TDX uptake was 2-fold higher in resistant MCFTDX cells than in parental MCF-7 cells, whereas in H630TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630TDX cells was noted when compared with the H630 parental cells. In H630TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCFTDX and H630TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.     

Expression and region-specific regulation of the oxytocin receptor gene in rat brain

The neuropeptide oxytocin (OT) exerts its various neurotransmitter functions via specific OT receptors (OTRs) that have been localized to distinct brain regions, including the ventromedial hypothalamus, the bed nucleus of stria terminalis, the amygdala, the subiculum, the hippocampus, and the olfactory nuclei. In the present study, we have characterized OTR gene expression by Northern blot and by semiquantitative RT-PCR in these brain regions and studied its regulation in response to estrogen (E2), progesterone, and the antiestrogen tamoxifen. We find that all regions analyzed express two messenger RNA (mRNA) bands (6.7 and 4.8 kb) that hybridize to a rat OTR complementary DNA probe and that correspond in size to two of the three OTR mRNA bands expressed in rat uterus. Analysis by RT-PCR, with two different primer pairs, did not reveal any structural differences between the coding regions of uterine and brain OTR mRNA. E2 treatment and gestation led to an 8-fold and a 6.5-fold increase in OTR mRNA levels, respectively. Progesterone was without effect, if administered alone, and did not influence the E2-induced rise in OTR mRNA. The E2 effect was restricted to E2-sensitive regions, such as the hypothalamus, and was not observed in the subiculum or the olfactory nuclei. Tamoxifen had a dual effect: on the one hand, it acted as a partial agonist in raising OTR mRNA levels in the hypothalamus of ovariectomized animals; on the other hand, it suppressed the E2-induced OTR mRNA rise in E2-sensitive brain regions. Although the present data do not exclude the possible existence of OTR subtype(s) in brain, they show that the uterine-type OTR gene is expressed in all major OTR-containing brain regions. Moreover, they show that region-specific regulation of OTR gene expression underlies the previously observed region-specific steroid regulation of central OT binding sites.     

Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence

Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.     

Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in reverse transcription-polymerase chain reaction

We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.     

Wilms' tumor suppressor gene (WT1) as a target gene of SRY function in a mouse ES cell line transfected with SRY

With the aim of identifying the gene(s) located downstream from SRY, we transfected an ES cell line with XX karyotype, TMA-18, with a Sry DNA construct and established cell lines, TS18-1 and TS18-2, where the transfected Sry was expressed in the functional linear mRNA form. Among the five potential SRY-target genes examined, i.e., MIS, SF1, P450arom, Sox9 and WT1, only the expression of WT1 was induced de novo by the unscheduled expression of Sry in the transfected cell lines. No clear indication of Sry-induced enhancement of Sox9 expression was obtained in the present series of experiments. Function of a yet unidentified gene(s) located on the Y chromosome might be needed for the up-regulation of Sox 9 expression which takes place during the development of male gonads. Quantitative RT-PCR analysis of the patterns of WT1 expression in developing fetal gonads revealed that although both male and female fetal gonads express WT1, male gonads invariably expressed WT1 mRNA at higher levels than female ones after the Sry expression. Immunohistochemical analysis of the male fetal gonads between 10.5 and 13.5 dpc demonstrated the presence of strong WT1 immunoreactivity in Sertoli cells of the primordial testes. Suggestions were made in the past indicating that both SF1 and WT1 proteins might be active in a common pathway upstream from Sry. Our results showed that WT1 is located downstream, rather than upstream from Sry and behaves independently from SF1. Analysis using an appropriate in vitro system will be essential to understand the molecular mechanisms of SRY action within cells.