Increased ErbB-2 Tyrosine Kinase Activity, MAPK Phosphorylation, and Cell Proliferation in the Prostate Cancer Cell Line LNCaP following Treatment by Select Pesticides

Daniel M. Tessier,1 and Fumio Matsumura

Department of Environmental Toxicology and the Center for Environmental Health Sciences, University of California, Davis, California 95616

Received September 12, 2000; accepted November 20, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oncogene erbB-2 codes for a receptor tyrosine kinase that functions as a key mitotic signal in a variety of cell types. Amplification or overexpression of erbB-2 occurs in many forms of cancer, such as of the breast, colon, and prostate, and is an indicator of poor prognosis in those diseases. In the human prostate cancer cell lines LNCaP and PC-3, erbB-2 kinase was activated by pesticides of different chemical classes: (1) the organochlorine insecticides ß-hexa-chlorocyclohexane (ß-HCH), o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT), and heptachlor epoxide; (2) the pyrethroid insecticide trans-permethrin, and (3) the fungicide chlorothalonil. o,p'-DDT also causes phosphorylation of mitogen-activated protein kinase (MAPK) and cellular proliferation of the androgen-dependent LNCaP line. However, no proliferative effect was observed in the androgen-independent PC-3 line. The proliferative effect of o,p'-DDT in LNCaP could not be blocked by the androgen receptor antagonist p,p'-dichlorodiphenyldichloroethene (p,p'-DDE), indicating that this effect of o,p'-DDT does not occur through direct interaction with the androgen receptor. Together these data demonstrate a putative mechanism for the action of certain pesticides in hormonal carcinogenesis.

Key Words: erbB-2/neu/HER-2; LNCaP; mitogen-activated protein kinase (MAPK); pesticides; prostate cancer; tyrosine kinases.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostate cancer is currently the second leading cause of cancer mortality, after lung cancer, in American men. Each year in the United States approximately 185,000 men are diagnosed with prostate cancer, and approximately 40,000 die from the disease (Landis, 1998). While the number of diagnosed cases has been rising for the past 30 years, our understanding of the causes and development of prostate cancer lags behind that of other common cancers, such as breast and colon cancers. Androgenic hormones influence the progression of prostate cancer during the early stages of the disease, when tumors are effectively managed by hormone ablation therapies. However, the resulting tumor regression is typically short-lived, and late-stage disease is aggressive, highly metastatic, and characterized by loss of hormonal regulation.

Environmental pollutants and dietary factors have been implicated in the incidence of prostate cancer (Giles and Ireland, 1997Go). The mechanisms by which environmental pollutants may initiate or promote prostate cancer are unknown. However, many environmental pollutants are known to interfere with hormonal regulation in hormone sensitive tissues (Colborn et al., 1993Go; Davis et al., 1993Go), which provides a framework for the action of endocrine disrupters in hormonal carcinogenesis (Huff et al., 1996Go). Organochlorine insecticides and their metabolites have been shown to antagonize the androgen receptor (for example, Kelce et al., 1995). Several epidemiological studies have identified an association between pesticide exposure and elevated rates of prostate cancer in farm workers and pesticide applicators (Blair and Zahm, 1995Go; Fleming et al., 1999Go; Mills, 1998Go; Morrison et al., 1993Go; Schreinemachers et al., 1999Go).

The oncogene erbB-2 codes for an epidermal growth factor (EGF)-receptor family tyrosine kinase (erbB-2, also known as HER-2 or c-neu) that is implicated in several types of cancer, including prostate and breast cancers. In the hormone-dependant prostate cancer cell line LNCaP, erbB-2 is overexpressed and functions in hormonally regulated mitotic signaling pathways (Meyers et al., 1996Go). Forced overexpression of erbB-2 kinase induces progression of prostate cancer cells in vitro, via the mitogen activated protein kinase (MAPK) signaling pathway (Yeh et al., 1999Go). MAPK signaling can confer hormone independence via a process of ligand-independent activation of the androgen receptor (Craft et al., 1999Go).

Our laboratory has previously reported increased erbB-2 kinase activity in hormone-responsive MCF-7 breast cancer cells following treatment by the organochlorine insecticides ß-hexa-chlorocyclohexane (ß-HCH), o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT), and other chlorinated compounds (Enan and Matsumura, 1998Go; Hatakeyama and Matsumura, 1999Go). In the present study we tested the ability of various pesticides to stimulate erbB-2 kinase activity in LNCaP cells and whether cellular proliferation was also stimulated. Test compounds were chosen based on the potential for human exposure (e.g., widespread use in agricultural practice and presence as low-level residue in food commodities), and as being representative of a range of chemical classes. Our results, together with results from MCF-7 breast cancer cells, indicate that stimulation of erbB-2 kinase activity by these compounds and subsequent cellular proliferation may be common phenomena in hormone-responsive cancer cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
Cell culture media and supplements were from Gibco BRL (Gaithersbuurg, MD). Fetal bovine serum, trypsin–EDTA solution, 5{alpha}-androstane-3{alpha},17ß-diol, testosterone, thiazolyl blue (MTT) and a tyrosine kinase substrate protein, random copolymer glutamic acid:tyrosine (EY 4:1, approximate molecular weight 40 KDa) was from Sigma (St. Louis, MO). ErbB-2 inhibitory antibody 9G6 was from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho- p42/44 MAPK antibody was from New England Biolabs, and ({gamma}-32-P)-ATP (3.0 µCi/mmol) was from Amersham (Piscataway, NJ). The pesticides alachlor (2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide), atrazine (6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine), captan (N-(trichloromethylthio)-4-cyclohexene-1,2-dicarboximide), carbendazim (2-benzimidazolecarbamic acid methyl ester), carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate), chlorothalonil (2,4,5,6-tetrachloro-1,3-benzene-dicarbonitrile), chlorpyrifos (phosphorothioic acid, O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) ester), cyhalothrin (3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylic acid cyano(3-phenoxyphenyl)methyl ester), 2,4-D (2,4-dichlorophenoxy)acetic acid), o,p'-DDT (o,p'-dichlorodiphenyltrichloroethane), diazinon (phosphorothioic acid, O,O-diethyl O-(6-methyl-2-(1-methylethyl)-4-pyrimidinyl) ester), ß-HCH (ß-hexachlorocyclohexane), trans-permethrin (3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methyl ester), trifluralin (2,6-dinitro-N,N-dipropyl-4-(trifluromethyl)benzenamine) were from Chem Service (Chester, PA).

Cell culture.
LNCaP and PC-3 human prostate cancer cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and grown in a 37°C, in 5% CO2 atmosphere. LNCaP cells were routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), streptomycin (100 units/ml) and amphotericin (0.25 µl/ml). PC-3 cells were routinely maintained in F-12 medium supplemented with 7% fetal bovine serum and glutamine, penicillin, streptomycin, and amphotericin, as above. In whole-cell chemical treatment experiments, fetal bovine serum in each of the respective media was heat-inactivated and treated with charcoal-dextran to remove lipophilic steroidal compounds. Cells were passaged by trypsin-EDTA treatment, and passage number was kept to less than 10 passages from ATCC stocks.

Preparation of postnuclear fraction.
Cells were grown to approximately 75% confluence in 100 mm diameter culture dishes. To prepare the postnuclear fraction, cells were rinsed twice in cold HEPES (50 mM, pH 7.5) and scraped in 1 ml homogenization buffer (50 mM HEPES pH 7.5, 1.5 mM KCl, 10 mM MgCl2, 2 mM DTT, 1 mM PMSF, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin A). The cell suspension was homogenized in a Dounce apparatus and centrifuged 12,000 x g for 10 min at 4°C. The supernatant was recovered as the postnuclear fraction. Aliquots were snap-frozen in liquid nitrogen and stored at –80°C.

Chemical treatment of postnuclear fraction.
The postnuclear fraction was adjusted to 1.0 mg/ml protein concentration. Aliquots of 200 µl were dosed with test compound dissolved in ethanol (vehicle concentration <= 1%) and incubated 10 min at ambient temperature. In antibody blocking experiments, postnuclear fraction was incubated with anti-erbB2 antibody 9G6 (0.1 µg) for 15 min prior to chemical treatment.

Protein tyrosine kinase assay.
Treated postnuclear fraction (protein concentration 1.0 mg/ml) was divided into 20 µl aliquots and the kinase reaction initiated by adding 15 µl reaction buffer, to result in final concentrations of 10 mM MnCl2, 10 µM Na3VO4, 0.25 µCi {gamma}-32P-ATP, and 1 µM radioinert ATP in 40 mM HEPES pH 7.5 containing 10 µg EY kinase substrate and 20 µg cellular protein. After incubation at 37°C for 5 min, the reaction was terminated by spotting 25 µl of the reaction mixture on phosphocellulose paper (Whatman P81). The paper was washed 3 times in phosphoric acid (85 mM), rinsed in acetone, and radioactivity measured by liquid scintillation counting.

MAP kinase activation assay.
LNCaP cells were grown to confluence in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cells were switched to stripped media (RPMI 1640 medium supplemented with 10% charcoal-dextran-treated fetal bovine serum) at 24 h and again at 2 h prior to addition of test chemicals. Test chemicals (EGF, 1 nM; o,p'-DDT, 100 nM) were added in stripped medium and incubated 10 min. Nuclear extracts of the treated cells were prepared for immunoblot analysis of phospo p44/42 MAP kinase. Cells were disrupted in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, PMSF, DTT, aprotinin, leupeptin, pepstatin A) and centrifuged at 3300 x g for 15 min. The nuclear pellet was recovered and washed twice in SHSG buffer (hypotonic buffer, 0.25 M sucrose, 0.5 % Triton X-100, 8.5 % glycerol, protease inhibitors) and once in SHS buffer (SHSG buffer minus detergents). The washed nuclear pellet was suspended in one volume buffer C (20 mM HEPES pH 7.9, 25% glycerol, 0.42 M NaCl, 0.5 mM EDTA, protease inhibitors) and incubated for 30 min on ice. The nuclear suspension was centrifuged for 15 min on an Eppendorf microfuge at maximum speed, and the supernatant was saved as the nuclear extract. Protein concentrations were determined and adjusted to 1.0 µg/µl. Nuclear proteins (25 µg) were resolved on a 10% SDS–PAGE gel and transferred to PVDF-plus membrane. The membrane was probed with anti-phospho p44/42 MAP kinase at 1:1000 dilution, followed by anti-rabbit IgG-HRP conjugate at 1:4000 dilution. Bands were visualized by chemiluminescence.

Proliferation assay.
Proliferation assays were conducted as described by Yeh et al. (1999). Cells were seeded into 24-well plates at 5 x 105 cells/well. The medium was changed to charcoal-dextran stripped medium after 24 h and test compound, dissolved in ethanol, was added. Control cells received vehicle only (ethanol). The treated cells were incubated for 72 h, the medium removed, and stripped medium containing MTT (thyazolyl blue, 1 mg/ml) was added. After 3-h incubation the medium was removed, and the formazan product solubilized in isopropanol. Absorbances were recorded at 540 nm.

Statistical analysis.
Experiments were run in triplicate or quadruplicate, and each experiment was repeated 2 or 3 times. Data from tyrosine kinase assays were compared by t-test of treated versus control samples. Proliferation assays were compared by one-way ANOVA. The criterion for significance was a p value < 0.05 for all comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosine Kinase Activation in Cell-Free System
The activation of protein tyrosine kinases by selected pesticides was determined in postnuclear fractions from LNCaP and PC-3 cells. As shown in Figure 1AGo, tyrosine kinase activity of LNCaP postnuclear fraction was significantly increased (t-test, p <= 0.05) by 100 nM concentrations of the fungicide chlorothalonil (230.7% of control), the organochlorine insecticides o,p-DDT (198.6%), heptachlor epoxide (180.4%), ß-HCH (243.6%) and the pyrethroid insectide trans-permethrin (235.9%). The fungicide captan resulted in a significant (t-test, p <= 0.05) decrease in tyrosine kinase activity (58.7% of control), while the remaining test compounds (trifluralin, 2,4-D, alachlor, chlorpyrifos, carbendazim, diazinon, cyhalathrin, atrazine, carbofuran) did not result in clear stimulatory or inhibitory effects. In PC-3 postnuclear fraction (Fig. 1BGo), tyrosine kinase activity was also significantly increased (t-test, p <= 0.05) by 100 nM concentrations of ß-HCH (182.1% of control), o,p'-DDT (133.5%) and trans-permethrin (130.6%).



View larger version (52K):
[in this window]
[in a new window]
 
FIG. 1. Activation of protein tyrosine kinases in LNCaP (A) and PC-3 (B) cell-free post-nuclear fractions by selected pesticides (all tested at 100 nM). Values are reported as % activity of ethanol-treated control and represent averages of 2 or 3 experiments run in triplicate. Asterisks indicate values statistically different from the control value (p <= 0.05, paired t-test).

 
To verify that the observed kinase activation is erbB-2-specific, LNCaP postnuclear fraction was preincubated with the erbB-2-specific inhibitory antibody 9G6. The increased kinase activity due to treatment by o,p'-DDT, trans-permethrin, and chlorothalonil was significantly reduced when erbB-2 kinase activity was inhibited by 9G6 antibody (Fig. 2Go). Preincubation with nonspecific antibody (mouse IgG) resulted in a slight decrease in kinase activity compared to control samples (i.e., no antibody), most likely due to nonspecific binding either of the kinases or substrate protein. It is therefore reasonable to conclude that either erbB-2 is the major tyrosine kinase effected by pesticide treatment, or it is the trigger molecule in a cascade of activated kinases in these experiments.



View larger version (60K):
[in this window]
[in a new window]
 
FIG. 2. Inhibition of erbB-2 tyrosine kinase activity by inhibitory antibody 9G6. LNCap postnuclear fraction was incubated with 9G6 or normal rabbit IgG prior to treatment with 100 nM o,p'-DDT, trans-permethrin (t-perm), or chlorothalonil (chthl). Values are reported as % activity of ethanol-treated control and represent averages of 2 or 3 experiments run in triplicate. Asterisks indicate values statistically different from the control value (p <= 0.05, paired t-test). PTK: protein tyrosine kinase.

 
Activation of MAP Kinase
The activation of mitogen-activated protein kinase (MAPK) by o,p'-DDT was studied in an LNCaP whole-cell system. Baseline levels of phosphorylated (i.e., activated form) of p42/44 MAPK were eliminated, prior to chemical treatment, by incubating cells in steroid hormone-depleted medium. Treatment with 100 nM o,p'-DDT resulted in elevated levels of p44/42 MAPK as determined by Western blot analysis using antibodies specific for the phosphorylated form of MAPK (Fig. 3Go). This activation occurred after a 40-min treatment, as compared to a more rapid response following exposure to the potent mitogen EGF (epidermal growth factor, used here as a positive control). The delayed response may indicate that o,p'-DDT must cross the cellular membrane to elicit its stimulatory effect. EGF, on the other hand, elicits a rapid response following its binding to an extracellular receptor site. Control cells treated with solvent carrier (ethanol) for 60 min did not show appreciable levels of activated MAPK. Therefore, the delayed response of MAPK activation shown following o,p'-DDT treatment is not due to the solvent effect or to background levels of endogenous growth factors.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 3. Immunoblot analysis showing the effect of o,p'-DDT on MAPK phosphorylation. Intact cells were treated for 10–60 min, lysed, and probed with antibodies specific for the phosphorylated form of p42/44 MAP kinase. EGF control cells were treated for 10 min; vehicle control cells were treated with ethanol for 60 min. The experiment was repeated 3 times with similar results. A representative blot is shown.

 
Cellular Proliferation
We next sought to determine if the kinase activation induced by agrochemical treatment was associated with effects occurring at the cellular level (i.e., in intact cells). When grown in steroid-depleted media, treatment of hormone-responsive LNCaP cells with o,p'-DDT resulted in a slight but significant (p <= 0.05) increase in cell proliferation (Fig. 4Go). The PC-3 cell line is commonly used in conjunction with the LNCaP line to distinguish androgen-dependent versus androgen-independent phenomena at the cellular level (Guo et al., 2000Go). When tested as intact cells under similar conditions of steroid-depleted growth, hormone-independent PC-3 cells were not effected by o,p-DDT treatment over a concentration range of 10 to 10,000 nM (data not shown).



View larger version (55K):
[in this window]
[in a new window]
 
FIG. 4. Proliferation of cultured LNCaP following treatment by the androgenic hormone dihydroxytestostrone (DHT) and the organochlorine insecticide o,p'-DDT. Cells were grown in steroid-depleted media and treated with test compound for 72 h prior to incubation with MTT. Values are the average of 2 experiments run in quadruplicate. Asterisks indicate values statistically different from the control value (p <= 0.05, ANOVA).

 
In a separate experiment (Fig. 5Go), we were able to show that the proliferative effect of o,p'-DDT in LNCaP cells could not be blocked by cotreatment with the antiandrogenic organochlorine compound p,p'-DDE, indicating that this effect of o,p'-DDT does not occur through direct interaction with the androgen receptor.



View larger version (70K):
[in this window]
[in a new window]
 
FIG. 5. Proliferation of cultured LNCaP following treatment by the organochlorine insecticide o,p'-DDT (100 nM) and the anti-androgenic DDT metabolite p,p'-DDE (100 nM). Cells were grown in steroid-depleted media and treated with test compound for 72 h prior to incubation with MTT. Values are the average of quadruplicate samples from two experiments. Asterisks indicate values statistically different from the control value (p <= 0.05, paired t-test). OD540, optical denisty at 540 nm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The erbB-2 receptor tyrosine kinase is overexpressed in human prostate, breast, and ovarian cancers, and is related to poor prognosis in those diseases (Yeh et al., 1999Go). The activation of erbB-2 by organochlorine compounds in the MCF-7 breast cancer system has previously been reported from our laboratory (Enan and Matsumura 1998Go, Hatakeyama and Matsumura 1999Go). In the present study we show that activation of erbB-2 by a broad range of compounds also occurs in the LNCaP and PC-3 cell lines, which are models for androgen-sensitive and androgen-insensitive prostate cells, respectively. Several lines of evidence indicate that the observed increase in kinase activity is the result of direct activation of erbB-2 tyrosine kinase rather than other types of interactions leading to activation of generic protein kinases: (1) The kinase assay utilized an artificial substrate containing only tyrosine residues as the site of phosphorylation. (2) The effect of chemical treatment on kinase activity could be blocked by an erbB-2-specific inhibitory antibody, 9G6. (3) The observed effects were shown in a cell-free postnuclear fraction in a short time span, eliminating the possibility of transcriptional effects. Immunoblot analysis of o,p'-DDT- and ß-HCH-treated cells verified that erbB-2 protein levels remain unchanged (data not shown). (4) Activation of isolated, immunoprecipitated erbB-2 from MCF-7 breast cancer cells by some of these chemicals has previously been demonstrated (Enan and Matsumura, 1998Go; Hatakeyama and Matsumura, 1999Go). Moreover, the observed effects on erbB-2 and MAPK occurred at a concentration (100 nM) that is physiologically relevant, given the known concentrations of some organochlorine insecticides in human tissue (Falck et al., 1992Go).

ErbB-2 stimulation by o,p'-DDT and ß-HCH was quantitatively greater in the prostate cell lines than was previously shown in the MCF-7 breast-cell line. These results in two distinct prostate cell lines indicate that the specific activation of this kinase is not unique to the MCF-7 system but may be a common phenomenon in erbB-2-expressing cancer cells. This specific interaction of 2 organochlorine pesticides with this important mitotic signaling protein led us next to determine if other pesticides could cause this same type of stimulation. It must be noted that we did not intend to perform a structure-activity relationship study, but rather a succinct survey of major-use pesticides, to determine the range of agrochemicals having the potential to stimulate erbB-2 in the same manner as o,p'-DDT and ß-HCH. Of the compounds tested, permethrin (a pyrethroid insecticide), chlorothalonil (a chlorothalonitrile fungicide) and heptachlor epoxide (an organochlorine insecticide metabolite) also stimulated erbB-2 kinase activity under cell-free conditions.

Given that erbB-2 is a major mitogenic signal protein in prostate-cancer cells, it was anticipated that whole-cell treatment with erbB-2 stimulating compounds would affect cellular proliferation. Interestingly, while erbB-2 was activated by chemical treatment in both the hormone-dependent LNCaP and hormone-independent PC-3 cell lines, increases in cellular proliferation was observed only in LNCaP. This suggests that the proliferative effect due to o,p'-DDT is somehow dependent upon the androgen receptor-signaling pathway, and consequently raises the question of whether the androgen receptor is directly stimulated. It has been shown previously that o,p'-DDT can competitively displace androgen from its receptor (Kelce et al., 1995Go) with an IC50 of approximately 100 µM. However, the effects we observed on erbB-2 activation occurred at 100 nM (3 orders of magnitude difference). Therefore erbB-2 kinase activation may be the more sensitive cellular response related to cellular proliferation. In support of this conclusion, we were able to show that the potent antiandrogen p,p'-DDE (IC50 = 5 µM), a direct antagonist of the androgen receptor (Kelce et al., 1995Go) could not block the proliferative effect of o,p'-DDT on LNCaP cells. The 20-fold difference in IC50s precludes significant displacement of p,p'-DDE by o,p'-DDT at the androgen receptor. Therefore the proliferative effect of o,p'-DDT in the presence of p,p'-DDE demonstrates that o,p'-DDT does not interact directly with the androgen receptor.

ErbB-2 kinase is known to activate unliganded androgen receptor in prostate cells (Craft et al., 1999Go). This is considered to represent one of many examples of "ligand independent activation" of hormone receptors (Weigel and Zhang, 1998Go). In a number of cases, the modulation of growth factor signal-mediating kinase or phosphatase activities are known to lead to activation of hormone receptors. In the case of extensively studied breast cancer cell lines, such as MCF-7, the activation of MAPK p42/44 by the growth factor receptor erbB-2 is the critical event mediating subsequent activation of the unliganded estrogen receptor. Although less is known about the mechanism of ligand-independent activation of the androgen receptor in prostate cancer cells, the relationship between MAPK activation and androgen receptor action has been observed in hormone-independent prostate cancer cell proliferation (Abreu-Martin et al., 1999Go). Androgen receptor target genes have been shown to be activated in the absence of androgen via an erbB-2/MAPK signal cascade (Yeh et al., 1999Go; Zhu and Liu, 1997Go). In LNCaP cells, heregulin, a natural activator of erbB-2 kinase (via the erbB-3 receptor) clearly activates MAPK (Grasso et al., 1997Go). In this cell line, erbB-2 is synergized by low levels of androgen. This evidence, and our experimental results, support our hypothesis that, a priori, the activation of the erbB-2 kinase signaling pathway induced by certain pesticides should elicit ligand-independent activation of the androgen receptor via activation of MAPK, leading to cellular proliferation in androgen receptor-rich LNCaP cells.


    ACKNOWLEDGMENTS
 
Marika Zai is acknowledged for technical assistance. This study was supported by research grants ES/CA 07284, ES05233, ES05707, and ES05887 from the National Institute of Environmental Health Sciences (NIEHS), NIH, Research Triangle Park, North Carolina.


    NOTES
 
1 To whom correspondence should be addressed at the University of Illinois at Chicago, School of Public Health (MC 922), Environmental and Occupational Health Sciences, 2121 West Taylor St., Chicago, IL 61612–7260. Fax: (312) 413-9898. Email: dmt{at}uic.edu. Back

The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abreu-Martin, M. T., Chari, A., Palladino, A. A., Craft, N. A., and Sawyers, C. L. (1999). Mitogen-activated protein kinase kinase kinase 1 activates androgen receptor-dependent transcription and apoptosis in prostate cancer. Mol. Cell. Biol. 19, 5143–5154.[Abstract/Free Full Text]

Blair, A., and Zahm, S. H. (1995). Agricultural exposures and cancer. Environ. Health Perspect. 103,(Suppl. 8), 205–208.[Medline]

Craft, N., Shostak, Y., Carey, M., and Sawyers, C. L. (1999). A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat. Med. 5, 280–285.[ISI][Medline]

Colborn, T., vom Saal, F. S., Soto, A. M. (1993). Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ. Health Perspect. 101, 378–384.[ISI][Medline]

Davis, L. D., Bradlow, H. L., Wolff, M., Woodruff, T., Hoel, D. G., and Anton-Culver, H. (1993). Medical hypothesis: Xenoestrogens as preventable causes of breast cancer. Environ. Health Perspect. 101, 372–377.[ISI][Medline]

Enan, E., and Matsumura, F. (1998). Activation of c-neu tyrosine kinase by o,p'-DDT and ß-HCH in cell-free and intact cell preparations from MCF-7 human breast cancer cells. J. Biochem. Mol. Toxicol. 12, 83–92.[Medline]

Falck, F., Jr, Ricci, A., Jr., Wolff, M. S., Godbold, J., and Deckers, P. (1992). Pesticides and poly-chlorinated biphenyl residues in human breast lipids and their relation to breast cancer. Arch. Environ. Health 47, 143–146.[ISI][Medline]

Fleming, L. E., Bean, J. A., Rudolph, M., and Hamilton, K. (1999). Cancer incidence in a cohort of licensed pesticide applicators in Florida. J. Occup. Environ. Med. 41, 279–288.[ISI][Medline]

Giles, G., and Ireland, P. (1997). Diet, nutrition and prostate cancer. Int. J. Cancer (Suppl.10), 13–17.

Guo, C., Luttrell, L. M., and Price, D. T. (2000). Mitogenic signaling in androgen-sensitive and -insensitive prostate cancer cell lines. J. Urol. 163, 1027–1032.[ISI][Medline]

Grasso, A. W., Wen, D., Miller, C. M., Rhim, J. S., Pretlow, T. G., and Kung, H. J. (1997). ErbB kinases and NDF signaling in human prostate cancer cells. Oncogene 15, 2705–2716.[ISI][Medline]

Hatakeyama, M., and Matsumura, F. (1999). Correlation between the activation of Neu tyrosine kinase and promotion of foci formation induced by selected organochlorine compounds in the MCF-7 model system. J. Biochem. Mol. Toxicol. 13, 296–302.[ISI][Medline]

Huff, J., Boyd, J., and Barrett, J. C. (1996). Hormonal carginogenesis and environmental influences: Background and overview. In Cellular and Molecular Mechanisms of Hormonal Carcinogenisis: Environmental Influences (J. Huff, J. Boyd, and J.C. Barrett, Eds.), pp. 3–22. Wiley-Liss, New York.

Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppainen, J. A., and Wilson, E. M. (1995). Persistent DDT metabolite p,p'-DDE is a potent androgen-receptor antagonist. Nature 375, 581–585.[ISI][Medline]

Landis, S. H., Murray, T., Bolden, S., and Wingo, P. A. (1998). Cancer Statistics. American Cancer Society, Atlanta.

Meyers, R. B., Oelschlager, D. K., Hockett, R. D., Rogers, M. D., Conway-Meyers, B. A., and Grizzle, W. E. (1996). The effects of dihydrotestosterone on the expression of p185erbB–2 and c-erbB-2 mRNA in the prostatic cell line LNCaP. J. Steroid Biochem. Molec. Biol. 59, 441–447.[ISI][Medline]

Mills, P. K. (1998). Correlation analysis of pesticide use data and cancer incidence rates in California counties. Arch. Environ. Health 53, 410–413.[ISI][Medline]

Morrison, H., Savitz, D., Semenciw, R., Hulka, B, Mao, Y., Morison, D., and Wigle, D. (1993). Farming and prostate cancer mortality. Am. J. Epidemiol. 137, 270–280.[Abstract]

Schreinemachers, D. M., Creason, J. P., and Garry, V. F. (1999). Cancer mortality in agricultural regions of Minnesota. Environ. Health Perspect. 107, 205–211.[ISI][Medline]

Weigel, N. L., and Zhang, Y. (1998). Ligand-independent activation of steroid hormone receptors. J. Molec.. Med. 76, 469–479.[ISI]

Yeh, S., Lin, H. K., Kang, H. Y., Thin, T. H., Lin, M. F., and Chang, C. (1999). From HER2/Neu signal cascade to androgen receptor and its coactivators: A novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells. Proc. Natl. Acad. Sci. U.S.A. 96, 5458–5463.[Abstract/Free Full Text]

Zhu, X., and Liu, J.P. (1997). Steroid-independent activation of androgen receptor in androgen-independent prostate cancer: A possible role for the MAP kinase signal transduction pathway? Mol. Cell. Endocrinol. 134, 9–14.[ISI][Medline]