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The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G(1) or G(2) phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways.

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), which is a critical step in glutathione biosynthesis. We isolated three BSO-resistant sublines, KB/BSO1, KB/BSO2, and KB/BSO3, from human epidermoid cancer KB cells. These cell lines showed 10-to 13-fold higher resistance to BSO, respectively, and had collateral sensitivity to cisplatin, ethacrynic acid, and alkylating agents such as melphalan and nitrosourea. Cellular levels of glutathione S-transferase pi (GST-pi) and its mRNA in BSO-resistant cell lines were less than 10% of the parental cells. Nuclear run-on assay showed that the transcriptional activity of GST-pi was decreased in BSO-resistant cells, and transient transfection of GST-pi promoter-chloramphenicol acetyltransferase constructs revealed that the sequences between -130 and -80 base pairs of the 5'-flanking region wer at least partially responsible for the decreased expression of the GST-pi gene. By contrast, gamma-GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the gamma-GCS gene was found to be amplified in the BSO-resistant cells lines. GST-pi mRNA levels appeared to be inversely correlated with gamma-GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-pi1 and KB/ BSO2-pi2, that overexpressed GST-pi, from KB/BSO3, after introducing a GST-pi expression plasmid. These two transfectants had similar levels in gamma-GCS mRNA, drug sensitivity to alkylating agents, and glutathione content at those of KB cells. These findings suggest that the cellular levels of GST-pi and gamma-GCS might be co-regulated in these novel BSO-resistant cells.

The nucleotide sequence of the mdr1 gene encoding a putative drug efflux pump (P-glycoprotein) is homologous to a class of bacterial membrane-associated transport proteins. These bacterial proteins are part of a multicomponent system that includes soluble periplasmic proteins that bind substrates, channeling them through the membrane in an energy-dependent manner. We have investigated the possibility that a similar multicomponent transport system exists in a multidrug-resistant human MCF-7 breast cancer cell line that was initially selected for resistance to doxorubicin (AdrR MCF-7). AdrR MCF-7 cells overexpress both the mdr1 gene and the pi class isozyme of glutathione S-transferase (GST-pi) (EC 2.5.1.18). The latter is one of several isozymes known to have a ligand-binding function in addition to drug-metabolizing capabilities. Although we have recently shown that transfection of a functional GST-pi expression vector is insufficient to confer resistance to doxorubicin in cells that lack P-glycoprotein expression [Mol. Pharmacol. 36:22-28 (1989)], we examined the possibility that GST-pi interacts with P-glycoprotein to alter multidrug resistance. To do this, we have cloned cDNAs encoding these proteins from AdrR MCF-7 cells, constructed expression vectors containing these two genes, and transfected these vectors sequentially into drug-sensitive MCF-7 cells. The human mdr1 cDNA isolated from AdrR MCF-7 is a variant gene whose sequence differs from that isolated previously from vinblastine-resistant KB cells [Cell 53:519-529 (1989)], resulting in an amino acid substitution of alanine to serine at position 893 (mdr1/893ala). Transfection of eukaryotic expression vectors containing the mdr1 gene isolated from AdrR MCF-7 cells produced a multidrug-resistant phenotype in recipient cells, with a cross-resistance pattern similar to that in the AdrR MCF-7 cells. To determine whether GST-pi expression could augment resistance provided by mdr1, two clones transfected with mdr1, one with high levels (153% of mdr1 RNA in AdR MCF-7 cells) and one with low levels (10% of mdr1 RNA in AdrR MCF-7 cells), were subsequently cotransfected with a GST-pi expression vector and pSVNeo and selected for resistance to G418. Six of these clones contained levels of GST-pi that were 8- to 18-fold greater than GST levels found in mdr1-expressing clones transfected with nonspecific DNA. We found no difference in the degree of resistance to doxorubicin, actinomycin D, and vinblastine between the clones expressing mdr1 only and the clones expressing both mdr1 and GST-pi.(ABSTRACT TRUNCATED AT 400 WORDS)

Racemic gossypol has been shown to have antitumor properties that may be due to its ability to uncouple tumor mitochondria or to its inhibitory effects on a variety of nonmitochondrial enzymes. We have studied the antimitochondrial and enzyme-inhibiting properties of gossypol in human carcinoma cell lines of breast (MCF-7, T47-D), ovarian (OVCAR-3) colon (HCT-8), and pancreatic (MiaPaCa) origin by comparing the effects of its purified (+)- and (-)-enantiomers. (-)-Gossypol shows up to 10-fold greater antiproliferative activity than (+)-gossypol in the cancer cell lines and in normal hematopoietic stem cells grown in vitro, with IC50 values ranging from 1.5 to 4.0 microM for the cancer cells and from 10 to 20 microM for the human marrow stem cells. As well, multidrug-resistant MCF/Adr cells appear more resistant to (-)-gossypol than their parental cell line. Electron microscopy indicates that the earliest ultrastructural change in tumor cells exposed to a cytotoxic (10 microM) concentration of (-)-gossypol is the selective destruction of their mitochondria. Consistent with this observation, 31P magnetic resonance spectroscopy detects pronounced changes in tumor cell high energy phosphate metabolism within 24 hr of (-)-gossypol treatment, manifest by 1.6- to greater than 50-fold differential reductions in the intracellular ratios of ATP/Pi, relative to (+)-gossypol-treated cell lines; the magnitude of these antimitochondrial effects correlates with the antiproliferative activity of (-)-gossypol. Northern blot RNA analyses suggest that treatment with a 5-10 microM dose of (-)-gossypol induces a transient increase in the expression of heat shock gene products, particularly hsp-70 transcripts. The mean 5-fold increase in (-)-gossypol-induced hsp-70 mRNA appears coincident with a comparable heat-stimulated increase in transcript levels, as compared with control or (+)-gossypol-treated cells. The enzyme-inhibiting properties of gossypol enantiomers were compared in cell-free assays measuring glutathione-S-transferase-alpha, -mu, and pi activities, calmodulin stimulation of cyclic nucleotide phosphodiesterase, and protein kinase C activity. Both enantiomers are near equivalent antagonists of calmodulin stimulation and protein kinase C activity, exceeding the potency of known inhibitors such as phenothiazines by as much as 50-fold. In contrast, (-)-gossypol is a 3-fold more potent inhibitor of glutathione-S-transferase-alpha and -pi isozyme activity, resulting in IC50 values of 1.6 and 7.0 microM, respectively, for these two isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Cultured wild-type MCF-7 human breast cancer cells and two MCF-7 sublines that overproduce enzymes of the de novo pyrimidine biosynthetic pathway were compared with regard to: rate of de novo biosynthesis of uracil nucleotides, sensitivity of the de novo and salvage pathways to the concentration of intracellular uracil nucleotides, and potential of exogenous uridine at concentrations equivalent to plasma levels to affect de novo pyrimidine biosynthesis. The PALAR MCF-7 subline, which is resistant to N-(phosphonacetyl)-L-aspartate and has 5.2 times the activity of the first de novo enzyme as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 5.8 times that of the wild type MCF-7 cells. The PYRR MCF-7 subline, which is resistant to pyrazofurin and has 15.1 times the activity of orotate phosphoribosyltransferase as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 1.4 times that of wild-type MCF-7 cells. These results are consistent with carbamyl phosphate synthetase being the rate-controlling step of de novo pyrimidine biosynthesis. In the presence of exogenous uridine at concentrations equivalent to that found in plasma (4.4-8.6 microM), the uracil nucleotide pool of wild-type MCF-7 cells was expanded by 20% and de novo synthesis was inhibited by 55%. Incubation of PALAR MCF-7 cells with uridine at concentrations between 7.3 and 16.8 microM caused a 40% increase in the uracil nucleotide pool and a 30% inhibition of de novo synthesis. De novo synthesis of uracil nucleotides in PYRR MCF-7 cells was not affected by a greater than 10-fold increase in the uracil nucleotide pool. Salvage of [14C] uridine was inhibited by an expanded uracil nucleotide pool in the wild-type and PYRR MCF-7 cells but was not inhibited in the PALAR MCF-7 cell line. These results demonstrate that, although the overproduced enzymes exhibit substrate affinities and specificities in cell-free preparations similar to those of the wild-type enzymes, in intact cells the resistant cell lines exhibit marked differences in the control of de novo and salvage pyrimidine biosynthetic pathways by intracellular uracil nucleotides.

The regulation of dihydrofolate reductase (DHFR) gene expression was studied in gene-amplified, estrogen-responsive human breast cancer cells (MTX MCF-7). Previous studies have shown that estrogen increases, whereas tamoxifen decreases the rate of DHFR enzyme synthesis resulting in corresponding changes in the level of this enzyme. DHFR levels also increase following incubation with methotrexate (MTX), an effect which is dependent on both the concentration of extracellular drug and the duration of exposure and which occurs at concentrations that are insufficient to inhibit cell growth. MTX, like estrogen and tamoxifen, has no apparent effect on the rate of DHFR enzyme degradation. The increase in DHFR in response to MTX is additive with that of estrogen and is not prevented by tamoxifen. Whereas hormone-mediated changes in DHFR are associated with changes in the level of DHFR mRNA, there is no apparent change in DHFR mRNA concentrations in cells exposed to MTX. The regulation of DHFR enzyme levels was also studied in gene-deleted Chinese hamster ovary cells which were transfected with a functional human DHFR minigene constructed from human DHFR genomic and cDNA sequences. Incubation with MTX increases DHFR levels in Chinese hamster ovary cells transfected with the human DHFR minigene but has no effect in cells transfected with a DHFR minigene which uses a viral promotor and polyadenylation signal. Thus, the human DHFR minigene contains sequences other than the protein coding region which effect the regulation of this gene by MTX.

MCF-7 human breast cancer cells, selected for resistance to adriamycin (AdrR), exhibit the phenotype of multidrug resistance (MDR). Previous studies have shown that resistance in AdrR MCF-7 cells is associated with several biochemical changes that are similar to those induced in rat hyperplastic nodules, preneoplastic liver lesions which display broad spectrum resistance to carcinogens and hepatotoxins. In this report, we show that these changes in the AdrR MCF-7 cells are also associated with the development of cross-resistance to the procarcinogen benzo(a)pyrene (BP) and are associated with a marked defect in the conversion of BP to its cytotoxic, carcinogenic metabolites by AdrR cells. Since aryl hydrocarbon hydroxylase is the principle enzyme activity which converts benzo(a)pyrene to toxic hydroxylated forms, the regulation of cytochrome P-450IA1 expression, the gene encoding this enzyme activity in MCF-7 cells, was examined. Incubation with 100 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h results in a marked increase in aryl hydrocarbon hydroxylase activity in wild type (WT) but not AdrR MCF-7 cells. The alteration in aryl hydrocarbon hydroxylase expression in the AdrR cells is not overcome by incubation either with higher concentrations of TCDD (1 microM) or for longer periods of time (4 days). Northern blot analysis indicates that this defect in AdrR MCF-7 cells involves a regulatory defect at the level of P-450IA1 RNA. Following transfection of a construct containing the normal mouse P-450IA1 promoter fused to a reporter gene (bacterial chloramphenicol acetyltransferase) into WT and AdrR MCF-7 cells, TCDD induced chloramphenicol acetyltransferase activity in WT MCF-7 cells only. Furthermore, TCDD also induces both DT-diaphorase and UDP-glucuronyltransferase activities in WT, but not AdrR cells. These data suggest that the defect in the AdrR MCF-7 cells is not due to a structural P-450IA1 gene mutation, but rather involves a product regulating the polycyclic hydrocarbon-inducible expression of several drug-metabolizing enzyme activities. This defect in the AdrR MCF-7 cells is also associated with the development of resistance to ellipticine, an anticancer agent which is converted to more toxic hydroxylated species by aryl hydrocarbon hydroxylase or a similar mixed function oxidase. The WT and AdrR MCF-7 cells represent a useful model to study the regulation of the P-450IA1 gene in human cells.

Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene-amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12%. Minigene-transfected cells contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFR gene sequences responsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected.

The cytosolic glutathione S-transferases (GSTs, EC 2.5.1.18) are a superfamily of dimeric isoenzymes which catalyze the conjugation of electrophilic substrates with glutathione. We have isolated from a murine cell line (L929) two mu class GST cDNAs, pmGT10 (1.4 kilobases (kb)) and pmGT2 (1.1 kb). Analysis of the deduced amino acid sequences revealed 93% homology between pmGT10 and the rat Yb1 (subunit 3) gene and 95% homology between pmGT2 and the rat Yb2 (subunit 4) gene. Furthermore, the 3′-untranslated regions of pmGT10 display a marked degree of homology to the 3′ region of the rat Yb1 gene, while this region of pmGT2 displays marked homology to the corresponding region of the rat Yb2 gene. Using probes specific for each gene, northern blot analysis demonstrated that pmGT10 and pmGT2 hybridized to a 1.3-1.4-kb mRNA species present in L929 cells. These two murine cDNAs were subcloned into bacterial expression vectors, and enzymatically active GSTs encoded by pmGT10 (mGTmu1) or pmGT2 (mGTmu2) were purified from transformed Escherichia colilysates. Western blot analysis of the individual GSTs produced in E. coliindicated that both genes encode GSTs which reacted with antibodies directed against mu class GST, but not with antibodies directed against alpha or pi class GSTs. The isoelectric points of these purified homodimers are 8.7 and 7.1, respectively, which are remarkably similar to those of the GST homodimers of the homologous rat subunits, 3-3 (or Yb1) and 4-4 (or Yb2), which are 8.4 and 6.9, respectively. Furthermore, the two murine subunits can form a heterodimer having a pI = 8.1 after in vitrodissociation with guanidine HCl, followed by dilution and dialysis to allow reassociation. Both homodimers of mGTmu1 and mGTmu2 and an apparent heterodimer are formed in L929 cells, as demonstrated by the isoelectric focusing profile of GST purified from this cell line. Hybridization of gene-specific probes with RNA from normal mouse tissues revealed that mGTmu1 transcripts were highly expressed in murine kidney, heart, lung, liver, and brain. Lower levels of mGTmu2 transcripts were also detected in kidney, heart, and lung. Expression of mGTmu2 RNA was not detected in murine liver or brain, which is in contrast to the regulation of the homologous rat subunit 4 (Yb2), which is expressed in liver and brain.

Adriamycin-resistant (AdrR) human breast cancer cells have been selected which exhibit cross-resistance to a wide range of anti-cancer drugs. This multidrug-resistant phenotype is associated with increases in the activities of glutathione peroxidase and glutathione transferase. The 45-fold increase in glutathione transferase activity is associated with the appearance of a new anionic isozyme in AdrR cells which is immunologically related to the anionic glutathione transferase present in human placenta. The increase in transferase and the level of drug resistance is relatively stable during passage of AdrR cells in the absence of adriamycin for over 10 months. A similar anionic glutathione transferase isozyme is also found in rat hyperplastic liver nodules, a preneoplastic state resulting from exposure to carcinogens. A rat cDNA which codes for the anionic glutathione transferase in rat hyperplastic nodules hybridizes to a 1.1-kilobase pair mRNA which is overexpressed in the AdrR MCF-7 cells. The anionic transferase has been purified from the AdrR cells and found to have characteristics which distinguish it from other anionic human glutathione transferases, including high levels of intrinsic peroxidase activity. The overexpression of a similar anionic glutathione transferase in human breast cancer cells selected for multidrug resistance and in rat hyperplastic liver nodules, which develop resistance to various hepatotoxins, suggests a possible role for this drug-conjugating enzyme in the mechanism of resistance in both of these states.

Methotrexate (MTX)-resistant human breast cancer cells (MTXR ZR-75) were obtained following serial passage of the wild-type ZR-75-1 cells (wild-type ZR-75) in MTX. The resistant cell line contains neither quantitative nor qualitative changes in dihydrofolate reductase compared to the parental line. Resistance is associated with a 3-fold decrease in MTX transport into MTXR ZR-75 cells as well as a 3-fold decrease in the activity of thymidylate synthetase in the resistant subline. Moreover, marked differences were observed between the wild-type and MTXR ZR-75 cells in their ability to convert MTX to its polyglutamate derivatives. Wild-type ZR-75 cells accumulate significant intracellular levels of antifolates during prolonged (24 h) exposure to 2 microM MTX, due to the formation of MTX polyglutamates. In contrast, essentially no polyglutamates are formed in the MTXR cells even during conditions which result in a vast excess of free intracellular drug in these cells. This defect is not associated with any apparent change in the activity of the enzyme folylpolyglutamyl synthetase, nor is there any alteration in the apparent Km of this enzyme for MTX in the resistant cells. Further studies demonstrate that the MTXR ZR-75 cells are cross-resistant to antifolate analogues which can be converted to polyglutamate derivatives (aminopterin and dichloromethotrexate), yet they are relatively sensitive to antifolate analogues such as 2,4-diamino-5-(3‘,4‘-dichlorophenyl)-6-methylpyrimidine, triazinate, and trimetrexate, which cannot be converted to polyglutamate forms. These studies identify a new mechanism (diminished accumulation of MTX polyglutamates) associated with resistance to MTX and lend additional support to the hypothesis that the formation of these derivatives is an important determinant of MTX cytotoxicity.

Methotrexate-resistant (MTXR) human breast cancer cells have been obtained which are 1000-fold less sensitive to this drug than the wild type MCF-7 cells from which they were derived. The resistant cells contain approximately a 25-fold increase in the activity of the target enzyme dihydrofolate (DHF) reductase. Enzyme inhibition studies and methotrexate affinity studies fail to reveal any difference in the DHF reductase present in the MTXR cells compared to wild type MCF-7 cells. Cytogenetic analysis demonstrates the presence of elongated marker chromosomes in the resistant cells which are not found in the parental cell line. Analysis of the DNA from MTXR and wild type MCF-7 cells using Southern blot hybridization indicates that the MTXR MCF-7 cells contain more copies of DHF reductase gene sequences than do wild type MCF-7 cells. These experiments also suggest that the amplified DHF reductase gene sequences in MTXR cells may have undergone a uniform structural rearrangement involving the 5' flanking sequences during the process of amplification. MTXR MCF-7 cells respond to estradiol by increasing cell growth, and the level of DHF reductase in the MTXR cells is further induced following administration of estradiol. Radiolabeling studies demonstrate that estrogen stimulates the actual synthesis of DHF reductase in human breast cancer cells.

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.

Increased expression of the glutathione S-transferase (GST; E.C.2.5.1.18) pi class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST pi and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalovirus immediate-early promoters. These GST pi expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST pi. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST pi. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumene hydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST pi. Southern blot analysis demonstrated the incorporation of the GST pi expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST pi RNA that was slightly larger than the endogenous GST pi RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5' leader sequence of the expression vector. The effect of increased GST pi activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-epoxide, as well as to ethacrynic acid (3.1-to 4.4-fold). Although increased GST pi expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)

A dihydrofolate reductase (DHFR) expression system composed of a DHFR minigene constructed from human DHFR genomic and cDNA sequences stably transfected into DHFR- Chinese hamster ovary cells was used to study the modulation of DHFR levels in response to release from amino acid deprivation. The addition of complete medium to cells grown for 48 hr in medium lacking isoleucine and glutamine caused the transfected cells to undergo a synchronous cycle of DNA replication. When DHFR protein levels assayed at the time of maximum DNA synthesis were compared to that present in the deprived state, levels rose 3.2- to 4.9-fold. By contrast, DHFR levels in cells transfected with a DHFR expression construct made from mouse DHFR cDNA fused to viral promoter, intervening, and polyadenylation sequences were not inducible under the identical conditions. Human DHFR minigene deletion or substitution constructs were used to determine which nucleotide sequences were responsible for amino acid-modulated expression. Although deletion of sequences upstream from 322 base pair 5' to the start of transcription did not affect DHFR expression, removal of sequences between 322 and 113 base pairs reduced DHFR induction by approximately 50%. Deletion of nucleotide sequences within the 3' nontranslated region of the gene also reduced the level of induction by approximately 50%. Reduction in the levels of DHFR RNA relative to total cellular RNA was also found. Thus, both 5' and 3' nucleotide sequences are involved in the modulation of DHFR levels following release from amino acid deprivation.

The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). We have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (AdrR MCF7), the anionic isozyme of glutathione S-transferase (GST pi). Hybridization with this GST pi cDNA, GST pi-1, demonstrated that increased GST pi activity in AdrR MCF7 cells is associated with overexpression but not with amplification of the gene. We mapped the GST pi gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GST pi overexpression are associated with the loss of ERs in AdrR MCF7 cells, we examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines we found an inverse association between GST pi expression and ER content. We also examined RNA from 21 primary breast cancers and found a similar association between GST pi expression and ER content in vivo. GST pi mRNA content in 11 ER-positive tumors (less than or equal to 10 fmol/mg of protein) was significantly different from the GST pi content of 10 ER-negative tumors (P = 0.002; Mann-Whitney Wilcoxon test for two independent samples). The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST pi than ER-positive tumors.

We have previously reported the isolation of a human breast cancer cell line resistant to doxorubicin (adriamycin; AdrR MCF-7 cells) that has also developed the phenotype of multidrug resistance (MDR). MDR in this cell line is associated with increased expression of mdr (P glycoprotein) gene sequences. The development of MDR in AdrR MCF-7 cells is also associated with changes in the expression of several phase I and phase II drug-detoxifying enzymes. These changes are remarkably similar to those associated with development of xenobiotic resistance in rat hyperplastic liver nodules, a well-studied model system of chemical carcinogenesis. Using an mdr-encoded cDNA sequence isolated from AdrR MCF-7 cells, we have examined the expression of mdr sequences in rat livers under a variety of experimental conditions. The expression of mdr increased 3-fold in regenerating liver. It was also elevated (3- to 12-fold) in several different samples of rat hyperplastic nodules and in four of five hepatomas that developed in this system. This suggests that overexpression of mdr, a gene previously associated with resistance to antineoplastic agents, may also be involved in the development of resistance to xenobiotics in rat hyperplastic nodules. In addition, although the acute administration of 2-acetylaminofluorene induced an 8-fold increase in hepatic mdr-encoded RNA, performance of a partial hepatectomy either before or after administration of 2-acetylaminofluorene resulted in a greater than 80-fold increase in mdr gene expression over that in normal untreated livers. This represents an important in vivo model system in which to study the acute regulation of this drug resistance gene.

The effect of human wild type and mutant p53 proteins on the human multidrug resistance (MDR1) promoter was studied in a p53-negative human cell line. Transient expression of MDR1 promoter-chloramphenicol acetyltransferase reporter gene constructs (MDRCAT) cotransfected with p53 expression vectors was analyzed in H358 lung carcinoma cells. Cotransfection with a wild type p53 expression vector stimulated MDRCAT activity, while cotransfection with mutant p53 expression vectors altered at amino acid positions 181, 252, 258, or 273 failed to stimulate expression. Wild type p53 stimulation of MDRCAT activity was time dependent with maximal expression occurring 24-30 h following transfection and correlating with high p53 protein levels. MDR1 promoter deletion analysis suggested that the sequences involved in wild type p53 stimulation of MDRCAT activity were contained within the region from -39 to +53 relative to the start of transcription at +1. This region contains no TATA or p53 consensus binding sequence but does contain an initiator sequence. Wild type p53 stimulation of MDRCAT expression also occurred in parental and doxorubicin-resistant SW620 colon and parental 2780 ovarian cancer cell lines, indicating that wild type p53-mediated simulation of the MDR1 promoter is not restricted to a single cell line.

MCF7 human breast cancer cells selected for resistance to doxorubicin (adriamycin; DoxR) have developed the phenotype of multidrug resistance. Multidrug resistance in DoxR MCF7 cells (called AdrR MCF7 cell line in previous publications) is associated with biochemical changes similar to those induced by carcinogens in rat hyperplastic liver nodules (HNs) and associated with resistance to xenobiotics in that system. In HNs and DoxR cells, exposure to a single agent results in the selection of cells that are cross-resistant to a wide variety of structurally dissimilar toxic agents. Resistance in both systems is associated with decreases in intracellular accumulation of toxins and changes in phase I (decreased cytochrome P1-450) and phase II (increased glutathione transferase and glucuronyltransferase) drug-metabolizing activities. In HNs and DoxR cells, resistance is associated with the induction of relatively stable levels of an immunologically related anionic glutathione transferase isozyme (EC 2.5.1.18). The finding of similar biochemical changes associated with the development of resistance to various xenobiotics in HNs and to many naturally occurring antineoplastic agents and at least one carcinogen (benzo[a]pyrene) in DoxR MCF7 cells suggests that the mechanisms of resistance in these two models may be similar.

Abnormalities in the p53 tumor suppressor gene have been shown to affect cellular processes related to cell cycle control and gene amplification. In this study we compare the status and function of wild-type p53 in MCF-7 breast cancer cells with sublines selected for resistance to chemotherapeutic agents having different mechanisms of action. Sublines that were resistant to melphalan, pyrazafurin, mitoxantrone, etoposide and PALA all retained expression of wild-type p53. Methotrexate-resistant MCF-7 cells were unusual heterozygotes that expressed a wild-type and dominant, in-frame p53 deletion mutant and the doxorubicin-resistant cells expressed only mutant p53. Analysis of the G1 checkpoint after treatment with ionizing radiation revealed that the pyrazafurin-, melphalan- and mitoxantrone-resistant cells arrested strongly in G1. The etoposide- and PALA-resistant cells had an intermediate G1 arrest phenotype and the methotrexate- and doxorubicin-resistant cells had a minimal G1 arrest phenotype. mRNA and protein analyses of downstream effector genes, including P21CIP1/Waf1, mdm2, Gadd 45 and the retinoblastoma protein, did not entirely differentiate sublines having a strong versus intermediate G1 arrest phenotype. Neither the p53 status nor the strength of the G1 arrest could be correlated with cell survival after ionizing radiation. When drug-sensitive MCF-7 cells were treated with the same chemotherapeutic agents, p53 and p21CIP1/Waf1 levels increased between 2- and 14-fold. Together these data suggest that other cellular factors likely play a role in overcoming the inhibitory effects of ionizing radiation on p53 in drug-resistant breast cancer cells.

Three antigenic variants of foot-and-mouth disease virus, type A, strain 119, were demonstrated in Ouchterlony analyses utilizing serum collected from guinea pigs 7 days postinfection (DPI). Such antisera contain antibodies of the 19S class. Guinea pig antisera that contained antibodies of the 7S class were unable to distinguish between the antigenic variants. Similarly, 19S antibody was able to demonstrate antigenic differences in trypsin- and chymotrypsin-treated viruses that were not detected by 7S antibody-containing antisera. One of the antigenic variants of virus is apparently the wild type and is tentatively considered to have two antigenic determinant groupings termed the a- and b-sites (140S-ab). The 140S-ab variant was the sole or predominant antigenic type produced in guinea pigs and in large plaque-forming- and tissue culture-low passage sources of the virus. Another antigenic variant appears to possess only the b-site (140S-b) and was the major constituent in tissue culture-high passage virus preparations. The third variant, a small plaque former, was also devoid of the a-site and contains an antigenic determinant that is related to, but not identical with, the b-site. This variant appears to be a minor constituent of tissue culture-high passage virus. 7-DPI serum could be absorbed with a suitable concentration of tissue culture-high passage virus to remove antibody reactive with the b-determinant site. This absorbed serum still precipitated 140S-ab virus by virtue of still containing antibody reactive with the a-determinant site; however, the neutralizing activity was eliminated. This suggests that the b-site is critical with respect to neutralization while the a-site is noncritical.

Radial immunodiffusion procedures have been developed for testing bovine sera for the presence of antibody activity against three foot-and-mouth disease virus types and a virus infection-associated antigen. Reactions with virus antigens were obtained as early as 4 days after infection, and the virus type causing the response was identified. The procedure had the advantages of great sensitivity for the detection of antibody and the ease with which antibodies to a variety of antigens could be detected.