Reactive oxygen species regulate properties of transformation in UROtsa cells exposed to monomethylarsonous acid by modulating MAPK signaling.

UROtsa cells exposed to 50 nM monomethylarsonous acid [MMA(III)] for 52 wk (MSC52) achieved hyperproliferation, anchorage independent growth, and enhanced tumorgenicity. MMA(III) has been shown to induce reactive oxygen species (ROS), which can lead to activation of signaling cascades causing stress-related proliferation of cells and even cellular transformation. Previous research established the acute activation of MAPK signaling cascade by ROS produced by MMA(III) as well as chronic up regulation of COX-2 and EGFR in MSC52 cells. To determine if ROS played a role in the chronic pathway perturbations by acting as secondary messengers, activation of Ras was determined in UROtsa cells [exposed to MMA(III) for 0-52 wk] and found to be increased through 52 wk most dramatically after 20 wk of exposure. Ras has been shown to cause an increase in O2(-) and be activated by increases in O2(-), making ROS important to study in the transformation process. COX-2 upregulation in MSC52 cells was confirmed by real time RT-PCR. By utilizing both antioxidants or specific COX inhibitors, it was shown that COX-2 upregulation was dependent on ROS, specifically, O2(-). In addition, because previous research established the importance of MAPK activation in phenotypic changes associated with transformation in MSC52 cells, it was hypothesized that ROS play a role in maintaining phenotypic characteristics of the malignant transformation of MSC52 cells. Several studies have demonstrated that cancer cells have lowered superoxide dismutase (MnSOD) activity and protein levels. Increasing levels of MnSOD have been shown to suppress the malignant phenotype of cells. SOD was added to MSC52 cells resulting in slower proliferation rates (doubling time=42h vs. 31h). ROS scavengers of OH also slowed proliferation rates of MSC52 cells. To further substantiate the importance of ROS in these properties of transformation in MSC52 cells, anchorage independent growth was assessed after the addition of antioxidants, both enzymatic and non-enzymatic. Scavengers of OH, and O2(-) blocked the colony formation of MSC52 cells. These data support the role for the involvement of ROS in properties of transformation of UROtsa cells exposed to MMA(III).

5-Aza-2'-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation.

The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2'-deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors.

The controlled and specific re-activation of endogenous tumor suppressors in cancer cells represents an important therapeutic strategy to block tumor growth and subsequent progression. Other than ectopic delivery of tumor suppressor-encoded cDNA, there are no therapeutic tools able to specifically re-activate tumor suppressor genes that are silenced in tumor cells. Herein, we describe a novel approach to specifically regulate dormant tumor suppressors in aggressive cancer cells. We have targeted the Mammary Serine Protease Inhibitor (maspin) (SERPINB5) tumor suppressor, which is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. Maspin is a multifaceted protein, regulating tumor cell homeostasis through inhibition of cell growth, motility and invasion. We have constructed artificial transcription factors (ATFs) made of six zinc-finger (ZF) domains targeted against 18-base pair (bp) unique sequences in the maspin promoter. The ZFs were linked to the activator domain VP64 and delivered in breast tumor cells. We found that the designed ATFs specifically interact with their cognate targets in vitro with high affinity and selectivity. One ATF was able to re-activate maspin in cell lines that comprise a maspin promoter silenced by epigenetic mechanisms. Consistently, we found that this ATF was a powerful inducer of apoptosis and was able to knock down tumor cell invasion in vitro. Moreover, this ATF was able to suppress MDA-MB-231 growth in a xenograft breast cancer model in nude mice. Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors.

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies.

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.

AP-2alpha and AP-2gamma are transcriptional targets of p53 in human breast carcinoma cells.

Activating enhancer-binding protein 2alpha (AP-2alpha) and activating enhancer-binding protein 2gamma (AP-2gamma) are transcription factors that bind GC-rich consensus sequences and regulate the expression of many downstream genes. AP-2alpha and AP-2gamma interact with p53 both physically and functionally. Expression microarray results in human breast carcinoma cells with forced p53 expression revealed AP-2gamma as a putative transcriptional target of p53. To confirm and extend these findings we measured the effects of forced p53 expression in human breast carcinoma cells by real-time reverse transcription-PCR, Western blotting, electrophoretic gel mobility shift assays, promoter reporter, chromatin immunoprecipitation and chromatin accessibility assays. Wild-type p53 expression rapidly induced not only AP-2gamma but also AP-2alpha mRNA. The subsequent increase in these proteins led to increased AP-2 DNA-binding and transactivating activity. Candidate p53-binding sites were identified in the AP-2alpha and AP-2gamma promoters. p53 binding to these cis-elements in vivo was also observed, together with a relaxation of chromatin structure in these regions. Finally, expression of either AP-2alpha or gamma inhibited growth of human breast carcinoma cells in vitro. Taken together, our findings indicate that these AP-2 genes are targets for transcriptional activation by p53 and suggest that AP-2 proteins may mediate some of the downstream effects of p53 expression such as inhibition of proliferation.

Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.

Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.

Transcriptional repression of BRCA1 by aberrant cytosine methylation, histone hypoacetylation and chromatin condensation of the BRCA1 promoter.

BRCA1 expression is repressed by aberrant cytosine methylation in sporadic breast cancer. We hypothesized that aberrant cytosine methylation of the BRCA1 promoter was associated with the transcriptionally repressive effects of histone hypoacetylation and chromatin condensation. To address this question, we developed an in vitro model of study using normal cells and sporadic breast cancer cells with known levels of BRCA1 transcript to produce a 1.4 kb 5-methylcytosine map of the BRCA1 5' CpG island. While all cell types were densely methylated upstream of -728 relative to BRCA1 transcription start, all normal and BRCA1 expressing cells were non-methylated downstream of -728 suggesting that this region contains the functional BRCA1 5' regulatory region. In contrast, the non-BRCA1 expressing UACC3199 cells were completely methylated at all 75 CpGs. Chromatin immunoprecipitations showed that the UACC3199 cells were hypoacetylated at both histones H3 and H4 in the BRCA1 promoter compared to non-methylated BRCA1 expressing cells. The chromatin of the methylated UACC3199 BRCA1 promoter was inaccessible to DNA-protein interactions. These data indicate that the epigenetic effects of aberrant cytosine methylation, histone hypoacetylation and chromatin condensation act together in a discrete region of the BRCA1 5' CpG island to repress BRCA1 transcription in sporadic breast cancer.

Changes in c-myc and c-fos expression in a human tumor cell line following exposure to bifunctional alkylating agents.

This study was initiated to determine if DNA-damaging chemotherapeutic agents can suppress the expression of oncogenes. The effects of three structurally related bifunctional alkylating agents on the steady state mRNA levels of c-myc, c-fos, N-ras, and beta-actin in the human colon carcinoma cell line Colo320HSR were examined. Colo320HSR has an amplified c-myc oncogene, which is highly overexpressed, and is assumed to be one of the transforming genes of this cell line. Two concentrations of mechlorethamine, L-phenylalanine mustard, and 4-hydroperoxycyclophosphamide, which produced 1 or 3 log cell kills were used to examine the effects of drug exposure on the expression of specific genes. Steady state mRNA levels were measured by Northern blot analysis. Following a 1-h drug exposure, RNA was isolated from cells at 0, 6, 12, and 24 h following drug removal. The agents used produced changes in the expression of specific genes, and all three did so in a similar fashion. Immediately following drug removal, the steady state expression of c-myc in treated cells was increased 2- to 3-fold compared to control. At 6 and 12 h following drug removal, c-myc levels were depressed 2.5- to 5-fold. By 24 h, c-myc expression approached, but remained below, control levels. Immediately following drug removal, c-fos levels were increased 3- to 4-fold, and from 6 to 24 h following drug removal, c-fos levels gradually return to, or fell below low basal levels. During the 24-h time course, drug treatment had little or no effect on the steady state levels of N-ras or beta-actin. These data support the hypothesis that alkylating agents may suppress the expression of specific transforming genes.

Collateral sensitivity to nitrosoureas in multidrug-resistant cells selected with verapamil.

We have examined the effects of the nitrosoureas, streptozotocin (STZ) and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), on a human multiple myeloma cell line, RPMI 8226, and its drug-resistant variants. Cell lines selected for doxorubicin (DOX) resistance alone displayed a STZ and BCNU cytotoxicity profile similar to that of the parent cell line. In contrast, two of the drug-resistant variants selected with DOX plus verapamil, an agent which inhibits P-glycoprotein-mediated multidrug resistance, displayed a collateral sensitivity to STZ and BCNU. Verapamil was included in the selection protocol because it has been shown to inhibit the P-glycoprotein-mediated multidrug resistance phenotype and is now in clinical trials as a chemosensitizing agent. The collateral sensitivity to these nitrosoureas seen in the DOX plus verapamil-selected cell lines is due to the functional loss of a DNA repair molecule, O6-Methylguanine DNA methyltransferase (MGMT). The functional loss of MGMT is secondary to the loss of MGMT gene expression. The loss of MGMT gene expression is not due to loss or gross rearrangement of the MGMT-coding region. If this selection pressure applied in vitro reflects the in vivo situation, then new chemotherapeutic strategies may be devised to exploit this phenomenon. These cell lines will serve as useful models for delineating mechanisms which govern MGMT expression.

Effects of streptozotocin/bis-chloroethylnitrosourea combination therapy on O6-methylguanine DNA methyltransferase activity and mRNA levels in HT-29 cells in vitro.

Treatment of chloroethylnitrosourea-resistant cells with streptozotocin (STZ) prior to bis-chloroethylnitrosourea (BCNU) exposure has been shown to result in a depletion of O6-methylguanine DNA methyltransferase (MGMT) activity, increased BCNU-induced interstrand cross-linking, and a 2-3 log enhancement of BCNU cytotoxicity in vitro. The current study was undertaken to define the kinetics of repletion of MGMT activity following the STZ/BCNU combination and to assess at the molecular level the effects of the combination on MGMT mRNA expression. Results demonstrate that MGMT activity can be depleted by greater than 90% relative to untreated controls using an optimized STZ/BCNU combination regimen and that greater than 50% depletion can be maintained for at least 24 h. This depletion appears to be independent of effects at the mRNA level because neither STZ alone nor the STZ/BCNU combination significantly altered steady state levels of MGMT mRNA. Cytotoxicity studies are consistent with MGMT repletion data and demonstrate that, as the interval between STZ and BCNU exposures increases, the degree of enhanced cytotoxicity induced by the combination relative to BCNU alone decreases. These results suggest that the enhanced cytotoxicity induced by the STZ/BCNU combination over BCNU treatment alone is favored by both the lack of induction of expression of MGMT mRNA and by slow reappearance of MGMT activity.

Analysis of multidrug resistance-associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells.

The multidrug resistance-associated protein (MRP) gene is a member of the ATP-binding cassette transporter gene superfamily and may be partially responsible for clinical drug resistance. Reverse transcriptase-polymerase chain reaction was used to measure MRP mRNA in normal hematopoietic cells from bone marrow and peripheral blood as well as patients with high risk acute myelocytic leukemia and multiple myeloma. All normal peripheral blood cells, regardless of cell lineage (CD4, CD8, CD14, CD15, CD19, CD56), expressed a similar basal level of MRP mRNA. Specimens from bone marrow containing mixed lineages also expressed a similar basal level of MRP expression. In patients with acute myelocytic leukemia, 10 of 12 (83%) of the specimens had detectable MRP mRNA, but the level of expression was similar to that of normal blood cells and low compared to a cell line known to overexpress MRP (H69/AR). All myeloma patients (12 of 12) had detectable MRP mRNA expression at levels comparable to normal peripheral blood and bone marrow cells. We conclude that MRP is commonly expressed in normal hematopoietic cells as well as certain hematopoietic malignancies. The therapeutic relevance of MRP expression is unknown, but these studies emphasize the importance of measuring MRP expression in normal cells as a point of reference and comparison for detection in malignant cells. We also recommend obtaining sequential specimens from patients, which may reveal an increased expression of MRP from baseline as the disease progresses and becomes resistant.

Evidence for cytoplasmic P-glycoprotein location associated with increased multidrug resistance and resistance to chemosensitizers.

A new human myeloma cell line, 8226/MDR10V, was selected from a P-glycoprotein-positive cell line, 8226/Dox40, in the continuous presence of doxorubicin and verapamil. MDR10V cells are 13-fold more resistant to doxorubicin and 4-fold more resistant to vincristine than the parent cell line, Dox40. Chemosensitizers are also less effective in reversing resistance in the MDR10V compared to the Dox40 cells. Despite higher resistance to cytotoxic agents, MDR10V expresses 40% less P-glycoprotein in the plasma membrane compared to Dox40; however, total cellular P-glycoprotein is the same in both cell lines. Confocal immunofluorescence microscopy shows 2.5-fold more P-glycoprotein in the cytoplasm of MDR10V cells as compared to Dox40 cells. The cytoplasmic location of P-glycoprotein in the MDR10V cells is associated with a redistribution of doxorubicin. In Dox40 cells, doxorubicin is concentrated in the nucleus, whereas in MDR10V cells, 90% of doxorubicin is found in the cytoplasm. In the presence of equivalent intracellular doxorubicin, there was a decrease in DNA-protein crosslinks in the MDR10V cell line compared to the Dox40 cell line. This finding is in agreement with the intracellular doxorubicin fluorescence studies showing less doxorubicin in the nuclei of MDR10V cells compared to Dox40 cells. Verapamil is less effective in increasing doxorubicin accumulation in the nuclei of MDR10V cells compared to Dox40 cells. Processing of P-glycoprotein from the endoplasmic reticulum to the medial Golgi was identical between the two cell lines as determined by endoglycosidase H sensitivity of newly sensitized P-glycoprotein. No mutations were found in MDR1 cDNA from MDR10V cells compared to Dox40 cells. These results suggest that resistance to chemosensitizing agents plus cytotoxic drugs is associated with a redistribution of P-glycoprotein from the plasma membrane to the cytoplasm, which in turn reduces the amount of cytotoxic drug reaching the nucleus.

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells.

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.

Semaphorin 7a exerts pleiotropic effects to promote breast tumor progression.

Understanding what drives breast tumor progression is of utmost importance for blocking tumor metastasis; we have identified that semaphorin 7a is a potent driver of ductal carcinoma in situ (DCIS) progression. Semaphorin 7a is a glycophosphatidylinositol membrane-anchored protein that promotes attachment and spreading in multiple cell types. Here, we show that increased expression of SEMA7A occurs in a large percentage of breast cancers and is associated with decreased overall and distant metastasis-free survival. In both in vitro and in vivo models, short hairpin-mediated silencing of SEMA7A reveals roles for semaphorin 7a in the promotion of DCIS growth, motility and invasion as well as lymphangiogenesis in the tumor microenvironment. Our studies also uncover a relationship between COX-2 and semaphorin 7a expression and suggest that semaphorin 7a promotes tumor cell invasion on collagen and lymphangiogenesis via activation of ß1-integrin receptor. Our results suggest that semaphorin 7a may be novel target for blocking breast tumor progression.

Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells.

BRCA1 mRNA is reduced in sporadic breast cancer cells despite the lack of mutations. Because a CpG island is found at the 5' end of the BRCA1 gene, we hypothesized that the decreased BRCA1 mRNA in sporadic breast cancer was associated with aberrant cytosine methylation of the CpG island. We examined BRCA1 mRNA expression in normal human mammary epithelial cells (HMECs), peripheral blood lymphocytes (PBLs) and six sporadic breast cancer cell lines using RT-PCR. The normal breast cells expressed high levels of BRCA1 mRNA. The sporadic breast cancer cell lines and PBLs expressed lower levels of BRCA1 mRNA ranging from a 3-16-fold decrease compared to the normal breast cells. We identified a 600 bp region of the BRCA1 CpG island that possessed strong promoter activity (approximately 40-fold above control), and determined the cytosine methylation patterns of the 30 CpG sites within this region by sodium bisulfite genomic sequencing. The HMECs, PBLs and five of the sporadic breast cancer cell lines were largely unmethylated. However, one sporadic breast cancer cell line, UACC3199, was > or = 60% methylated at all 30 CpG sites (18 sites were 100% methylated) and was associated with an eightfold decrease in BRCA1 mRNA compared to normal breast cells. These findings suggest that aberrant cytosine methylation of the BRCA1 CpG island promoter may be one mechanism of BRCA1 repression in sporadic breast cancer.

Analysis of the Harvey ras gene in cisplatin-initiated mouse skin tumors by polymerase chain reaction and direct DNA sequencing.

Cisplatin (cis-dichlorodiammineplatinum (II)) acts as a tumor initiator in the mouse skin model of carcinogenesis. DNA transfection studies suggested that skin tumors initiated by cisplatin contained dominant transforming activity. Since the Harvey-ras (H-ras) gene is known to be activated by point mutations in chemically initiated mouse skin tumors, we used polymerase chain reaction (PCR) and direct DNA sequencing to analyze the DNA sequence of the H-ras gene in twelve different cisplatin-initiated skin tumors. The results of these studies indicated that cisplatin-initiated skin tumors were normal (wild-type) at codons 12, 13, 61 and 117. Thus the transforming activity associated with cisplatin initiated mouse skin tumors was not due to a mutant H-ras gene and this suggests the involvement of other transforming genes during initiation of the mouse skin with cisplatin.

Analysis of MRP mRNA in mitoxantrone-selected, multidrug-resistant human tumor cells.

MRP, a gene recently isolated from a non-P-glycoprotein-mediated multidrug-resistant small cell lung cancer cell line, is a candidate multidrug-resistance gene. Mitoxantrone, an anthracenedione antitumor agent, frequently selects for non-P-glycoprotein-mediated multidrug resistance in in vitro models. To determine whether mitoxantrone-selected multidrug resistance was due to overexpression of MRP, we examined the expression of MRP in four mitoxantrone-selected, multidrug-resistant human tumor cell lines, using a reverse transcriptase/polymerase chain reaction assay. Results from these experiments suggest that overexpression of MRP does not appear to play a primary role in mitoxantrone-selected multidrug resistance in these cell lines, and that other novel drug-resistance mechanisms are likely.

Developmental expression of N-acetyltransferases in C57BI/6 mice.

N-Acetyltransferases (NATs) play an important role in the biotransformation of a wide variety of arylamine drugs and carcinogens. Two genes (NAT1, NAT2) have been identified and allelic variation in NAT2 has been associated with arylamine toxicity in adults. Little information has been reported on expression of NAT genes during embryonic and fetal development although substrate specific NAT activity has been detected. The current study investigated the expression of NAT1 and NAT2 in mice pre-and postnatally. RNA was isolated from maternal liver, embryonic tissue at gestational days (GD) 10, 15, and 18, or neonates at neonatal day 3. Reverse transcription-polymerase chain reaction was performed using primers designed to amplify portions of either the NAT1 or the NAT2 gene. NAT1 and NAT2 mRNAs were detected in the embryo/placental complex at GD 10 and in GD 15 and 18 embryos. NAT2 but not NAT1 was expressed in GD 18 and neonatal day 3 hepatic tissue. These data demonstrate the differential expression of NAT genes in the mouse embryo and suggest a potential role for NAT in development.