The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.
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We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites. (+info)
Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells.
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DNA-dependent protein kinase (DNA-PK) functions in double-strand break repair and immunoglobulin [V(D)J] recombination. We previously established a radiation-sensitive human cell line, M059J, derived from a malignant glioma, which lacks the catalytic subunit (DNA-PKcs) of the DNA-PK multiprotein complex. Although previous Northern blot analysis failed to detect the DNA-PKcs transcript in these cells, we show here through quantitative studies that the transcript is present, albeit at greatly reduced (approximately 20x) levels. Sequencing revealed no genetic alteration in either the promoter region, the kinase domain, or the 3' untranslated region of the DNA-PKcs gene to account for the reduced transcript levels. Nuclear run-on transcription assays indicated that the rate of DNA-PKcs transcription in M059J and DNA-PKcs proficient cell lines was similar, but the stability of the DNA-PKcs message in the M059J cell line was drastically (approximately 20x) reduced. Furthermore, M059J cells lack an alternately spliced DNA-PKcs transcript that accounts for a minor (5-20%) proportion of the DNA-PKcs message in all other cell lines tested. Thus, alterations in DNA-PKcs mRNA stability and/or the lack of the alternate mRNA may result in the loss of DNA-PKcs activity. This finding has important implications as DNA-PKcs activity is essential to cells repairing damage induced by radiation or radiomimetric agents. (+info)
Collagenase-3 (MMP-13) is expressed by tumor cells in invasive vulvar squamous cell carcinomas.
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Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by invading tumor cells in squamous cell carcinomas (SCCs) of the head and neck. Here, we have further elucidated the role of MMP-13 in tumor invasion by examining its expression in invasive malignant tumors of the female genital tract. Using in situ hybridization, expression of MMP-13 mRNA was detected in 9 of 12 vulvar SCCs, primarily in tumor cells, but not in intact vulvar epithelium, in cervical SCCs (n = 12), or in endometrial (n = 11) or ovarian adenocarcinomas (n = 8). MMP-13 expression was especially abundant in vulvar carcinomas showing metastasis to lymph nodes and was associated with expression of membrane type 1 MMP by tumor cells and gelatinase-A (MMP-2) by stromal cells, as detected by immunohistochemistry. MMP-13 mRNAs were detected in 9 of 11 cell lines established from vulvar carcinomas and in 4 of 6 cell lines from cervical carcinomas, whereas endometrial (n = 10) and ovarian (n = 9) carcinoma cell lines were negative for MMP-13 mRNA. No correlation was detected between MMP-13 expression and p53 gene mutations in vulvar SCC cell lines. However, MMP-13 expression was detected in 5 of 6 vulvar and cervical SCC cell lines harboring HPV 16 or 68 DNA. These results show that MMP-13 is specifically expressed by malignantly transformed squamous epithelial cells, including vulvar SCC cells, and appears to serve as a marker for their invasive capacity. (+info)
Specific chromosomal aberrations and amplification of the AIB1 nuclear receptor coactivator gene in pancreatic carcinomas.
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To screen pancreatic carcinomas for chromosomal aberrations we have applied molecular cytogenetic techniques, including fluorescent in situ hybridization, comparative genomic hybridization, and spectral karyotyping to a series of nine established cell lines. Comparative genomic hybridization revealed recurring chromosomal gains on chromosome arms 3q, 5p, 7p, 8q, 12p, and 20q. Chromosome losses were mapped to chromosome arms 8p, 9p, 17p, 18q, 19p, and chromosome 21. The comparison with comparative genomic hybridization data from primary pancreatic tumors indicates that a specific pattern of chromosomal copy number changes is maintained in cell culture. Metaphase chromosomes from six cell lines were analyzed by spectral karyotyping, a technique that allows one to visualize all chromosomes simultaneously in different colors. Spectral karyotyping identified multiple chromosomal rearrangements, the majority of which were unbalanced. No recurring reciprocal translocation was detected. Cytogenetic aberrations were confirmed using fluorescent in situ hybridization with probes for the MDR gene and the tumor suppressor genes p16 and DCC. Copy number increases on chromosome 20q were validated with a probe specific for the nuclear receptor coactivator AIB1 that maps to chromosome 20q12. Amplification of this gene was identified in six of nine pancreatic cancer cell lines and correlated with increased expression. (+info)
Detection of occult lymph node metastases in esophageal cancer by minimally invasive staging combined with molecular diagnostic techniques.
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BACKGROUND AND OBJECTIVES: Lymph node metastases are the most important prognostic factor in patients with esophageal cancer. Histologic examination misses micrometastases in up to 20% of lymph nodes evaluated. In addition, non-invasive imaging modalities are not sensitive enough to detect small lymph nodes metastases. The objective of this study was to investigate the use of reverse transcriptase-polymerase chain reaction (RT-PCR) of messenger RNA (mRNA) for carcinoembryonic antigen (CEA) to increase the detection of micrometastases in lymph nodes from patients with esophageal cancer. METHODS: RT-PCR of CEA mRNA was performed in lymph nodes from patients with malignant and benign esophageal disease. Each specimen was examined histopathologically and by RT-PCR and the results were compared. RESULTS: Metastases were present in 29 of 60 (48%) lymph nodes sample by minimally invasive staging from 13 patients with esophageal cancer when examined histopathologically. RT-PCR identified nodal metastases in 46 of these 60 (77%) samples. RT-PCR detected CEA mRNA in all 29 histologically positive samples and in 17 histologically negative lymph nodes. All lymph nodes from patients with benign disease (n = 15) were negative both histopathologically and by RT-PCR. The stage of two patients was reclassified based on the RT-PCR results, which identified lymph node spread undetected histopathologically. Both of these patients developed recurrent disease after resection of the primary tumor. CONCLUSIONS: RT-PCR is more sensitive than histologic examination in the detection of lymph node metastases in esophageal cancer and can lead to diagnosis of a more advanced stage in some patients. The combination of minimally invasive surgical techniques in combination with new molecular diagnostic techniques may improve our ability to stage cancer patients. (+info)
Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells.
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Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases alpha, delta and epsilon), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)-adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5-4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radio-labelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2-4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3-4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16-40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 microM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle. (+info)
Improved methods using the reverse transcriptase polymerase chain reaction to detect tumour cells.
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Reverse transcriptase polymerase chain reaction (RT-PCR) is increasingly used to detect small numbers of circulating tumour cells, though the clinical benefit remains controversial. The largest single contributing factor to the controversy of its value is the different approaches to sample processing. The aim of this study was to compare the sensitivity and reproducibility of RT-PCR for the detection of tumour cells after four commonly used different methods of sample processing. Using RT-PCR, one tumour cell spiked in 2 ml of whole blood was detected after analysis of separated mononuclear cell RNA, whole blood total or poly-A+ RNA. No false positives were identified with any method. However, the reproducibility of tumour cell detection was reduced after isolation of the mononuclear cell fraction. Only analysis of poly-A+ RNA had a sensitivity of 100% in all the cell spiking experiments. In patient blood samples, analysis of poly-A+ RNA increased the number of blood samples positive for tyrosine hydroxylase (TH) mRNA compared with those positive after analysis of total RNA. This may reflect high levels of cDNA reducing the efficiency of the PCR. Isolation of poly-A+ RNA increases the sensitivity and reproducibility of tumour cell detection in peripheral blood. (+info)
Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines.
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BACKGROUND: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein). Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines. This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone. METHODS: Total cellular RNA or poly A+ RNA and genomic DNA were isolated from parental and drug-selected cell lines. Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively. RESULTS: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma (S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079), and myeloma (8226) origins. Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells. CONCLUSIONS: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug. It is likely that BCRP is the putative "mitoxantrone transporter" hypothesized to be present in these cell lines. (+info)