Biomedical subjects

L J Hafer

Publications and source records attributed to L J Hafer.

8 recordsLinked to original sources

PubMed2001

Immunodetection of nmt55/p54nrb isoforms in human breast cancer.

BACKGROUND: We previously identified and characterized a novel 55 kDa nuclear protein, termed nmt55/p54nrb, whose expression was decreased in a subset of human breast tumors. The objective of this study was to determine if this reduced expression in human breast tumors was attributed to the regulation of mRNA transcription or the presence of altered forms of this protein. RESULTS: Northern blot analysis and ribonuclease protection assay indicated that nmt55/p54nrb mRNA is expressed at varying levels in estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast tumors suggesting that reduced expression of nmt55/p54nrb protein in ER- tumors was not due to transcriptional regulation. To determine if multiple protein isoforms are expressed in breast cancer, we utilized Western blot and immunohistochemical analyses, which revealed the expression of an nmt55/p54nrb protein isoform in a subset of ER+ tumors. This subset of ER+ human breast tumors expressed an altered form of nmt55/p54nrb that was undetectable with an amino-terminal specific antibody suggesting that this isoform contains alterations or modifications within the amino terminal domain. CONCLUSIONS: Our study indicates that nmt55/p54nrb protein is post-transcriptionally regulated in human breast tumors leading to reduced expression in ER- tumors and the expression of an amino terminal altered isoform in a subset of ER+ tumors. The potential involvement of nmt55/p54nrb in RNA binding and pre-mRNA splicing may be important for normal cell growth and function; thus, loss or alteration of protein structure may contribute to tumor growth and progression.

Adenocarcinoma↗

PubMed2001

Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture.

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2000

Expression of the aryl hydrocarbon receptor/transcription factor (AhR) and AhR-regulated CYP1 gene transcripts in a rat model of mammary tumorigenesis.

Exposure to ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons (PAH), may contribute to human breast cancer. In animals, PAH induce tumors in part by activating the aryl hydrocarbon receptor (AhR)/transcription factor. Historically, investigations into AhR-regulated carcinogenesis have focused on AhR-dependent transcriptional regulation of cytochrome P450 (CYP) enzymes which oxidize PAH to mutagenic intermediates. However, recent studies suggest that the AhR directly regulates cell growth. Given the postulated role of the AhR in carcinogenesis, we predicted that: (1) tissue predisposed to PAH tumorigenesis would express the AhR and (2) aberrant AhR and/or AhR-regulated gene expression would accompany malignant transformation. To test these hypotheses, AhR and CYP1 protein and/or mRNA levels were evaluated in rat mammary tumors induced with 7, 12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH and AhR ligand. Results indicate modest AhR expression in normal mammary myoepithelial and ductal epithelial cells. In contrast, high AhR levels were detected in DMBA-induced tumors. Nuclear AhR localization in tumors suggested constitutive AhR activation. In situ hybridization and quantitative RT-PCR assays indicated high AhR mRNA levels in neoplastic epithelial cells. While both AhR-regulated CYP1A1 and CYP1B1 mRNAs were induced in breast tissue within 6 h of DMBA gavage, only CYP1B1 mRNA remained elevated in tumors. These results: (1) help explain targeting of breast tissue by carcinogenic PAH, (2) imply that AhR and CYP1B1 hyper-expression represent molecular biomarkers for, at least, PAH-induced mammary cell transformation, and (3) suggest mechanisms through which the AhR may contribute to carcinogenesis well after exogenous AhR ligands have been eliminated.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2000

Activation of NF-kappaB/Rel occurs early during neoplastic transformation of mammary cells.

NF-kappaB/Rel is a family of transcription factors which are expressed in all cells; however, in most non-B cells, they are sequestered in the cytoplasm in inactive complexes with specific inhibitory proteins, termed IkappaBs. We have recently shown that NF-kappaB/Rel factors are aberrantly activated in human breast cancer and rodent mammary tumors, and function to promote tumor cell survival and proliferation. Here, we have examined the time-course of induction of NF-kappaB/Rel factors upon carcinogen treatment of female Sprague-Dawley (S-D) rats in vivo and in human mammary epithelial cells (HMECs) in culture. We observed that NF-kappaB/Rel activation is an early event, occurring prior to malignant transformation. In S-D rats, increased NF-kappaB/Rel binding was detected in nuclear extracts of mammary glands from 40% of animals 3 weeks post-treatment with 15 mg/kg 7,12-dimethylbenz[a]anthracene (DMBA); this is prior to formation of tumors which normally begin to be detected after 7-9 weeks. In non-tumorigenic MCF-10F cells, in vitro malignant transformation upon treatment with either DMBA or benzo[a]pyrene (B[a]P) resulted in a 4- to 12-fold increase in activity of classical NF-kappaB (p65/p50). NF-kappaB induction was corrrelated with a decrease in the stability of the NF-kappaB-specific inhibitory protein IkappaB-alpha. Ectopic expression of the transactivating p65 subunit of NF-kappaB in MCF-10F cells induced the c-myc oncogene promoter, which is driven by two NF-kappaB elements, and endogenous c-Myc levels. Furthermore, reduction mammoplasty-derived HMECs, immortalized following B[a]P exposure, showed dysregulated induction of classical NF-kappaB prior to malignant transformation. Together these findings suggest that activation of NF-kappaB plays an early, critical role in the carcinogen-driven transformation of mammary glands.

Animals↗

PubMed1999

Dietary fat, body weight, and cancer: contributions of studies in rodents to understanding these cancer risk factors in humans.

Understanding diet and energy balance as risk factors for breast, colon, and other cancers requires information on the contribution of each factor and of interactions among factors to cancer risk. Rodent models for breast cancer provide extensive data on effects of dietary fat and calories, energy balance, body weight gain, and physical activity on tumor development. Analyses of the combined data from many studies have shown clearly that quality and quantity of dietary fat and energy balance contribute independently to increased mammary gland tumorigenesis. These findings were seen in female rats fed diets high in fat (35-40% of calories) compared to rats fed control diets, with approximately 10% of calories as fat (Fay and Freedman, 1997, Breast Cancer Res. Treat. 46, 215-223). The methods used permit comparison of experimental and epidemiological data, and they may be useful in extrapolating between species and developing public health recommendations. In addition to the contributions of lifetime-diet composition, intake, energy balance, and physical activity to cancer risk, there are questions about the timing and duration of alterations in these factors and about the "dose-response" characteristics of cancer risk to the factors. Endocrine mechanisms may be significant in mammary gland tumor risk, but experimental and epidemiological data indicate that cancers at other sites, such as colon and liver, also are influenced by the factors listed. Other diet and lifestyle factors that influence energy, or specifically fat, metabolism may also affect risk for cancers that are promoted by increased intake of fat and calories. Studies of separate and interactive effects of dietary fat, black tea, weight gain, and mammary gland tumorigenesis (Rogers, et al, 1998, Carcinogenesis 19, 1269-1273) have been analyzed. Using adjustment of carcinogenesis endpoints for body weight, tumor burden, and latency, they were found to be related to weight gain within treatment groups in 2 of 3 experiments.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

Black tea and mammary gland carcinogenesis by 7,12-dimethylbenz[a]anthracene in rats fed control or high fat diets.

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor-bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

Induction of PreB cell apoptosis by 7,12-dimethylbenz[a]anthracene in long-term primary murine bone marrow cultures.

Numerous studies demonstrate that polycyclic aromatic hydrocarbons (PAH) suppress immunity by modifying the function of both B and T cells. Relatively few studies have assessed the effects of these common environmental chemicals on immature lymphocytes. In the present study, long-term primary bone marrow cultures were employed to investigate the effects of a prototypic PAH and aryl hydrocarbon receptor (AhR) agonist, 7,12-dimethylbenz[a]anthracene (DMBA), on immature B lymphocytes. In this system, immature preB cells are maintained in a supportive microenvironment provided by bone marrow stromal cells. Results presented here demonstrate that (1) exposure of primary bone marrow cultures to DMBA results in preB cell death by apoptosis; (2) notably low doses of DMBA (> or = 10(-8) M) induce preB cell apoptosis; (3) in long-term cultures, bone marrow stromal cells, but not preB cells, express AhR mRNA and protein as determined by in situ hybridization, RT-PCR, and immunoblotting; (4) freshly isolated unfractionated bone marrow cells, but not purified bone marrow B cells, express AhR protein as assessed by immunohistochemistry; (5) alpha-naphthoflavone, a competitive AhR inhibitor and cytochrome P450 antagonist, completely blocks DMBA-induced preB cell apoptosis in primary bone marrow cultures; and (6) DMBA or benzo[a]pyrene injection in vivo results in bone marrow cell apoptosis consistent with the death of hematopoietic cells clustered around stromal elements. The results implicate programmed cell death as a mechanism underlying DMBA-mediated immunosuppression and suggest that preB cell death is influenced by local interactions with AhR+ bone marrow stromal cells.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1992

CA-549 is not diagnostically useful as an immunohistochemical marker for breast carcinoma.

We evaluated BC4E549, a monoclonal antibody to CA-549, for use as an immunohistochemical marker to determine (1) differences in staining patterns between breast carcinoma and nonmalignant breast tissue specimens, (2) sensitivity and specificity of staining patterns for CA-549 in breast carcinoma vs other carcinomas, and (3) conservation of primary staining patterns in tumor metastases. We studied paraffin-embedded tissue specimens from 382 cases including nonmalignant breast tissue, endometrium, lung, pancreas, and thyroid gland; breast carcinoma; and adenocarcinomas of the colon, endometrium, lung, ovary, pancreas, and thyroid gland. Membranous staining was present in all nonmalignant breast tissue specimens and in most breast carcinoma tissue specimens. Diffuse cytoplasmic staining was more extensive in breast carcinomas than in benign processes. However, similar staining was also seen in normal and neoplastic tissue specimens obtained from a variety of other sites. Sensitivity and specificity of diffuse cytoplasmic staining for CA-549 in breast carcinoma relative to carcinomas from other sites studied were 78.6% and 77.4%, respectively. Staining patterns were not preserved from primary tumor to metastases in 67.4% of cases evaluated. We conclude that BC4E549 antibody to CA-549 is not a sufficiently sensitive or specific immunohistochemical marker in routinely processed tissue specimens to be useful in the clinical-diagnostic laboratory setting.

Antibodies, Monoclonal↗