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Biomedical subjects

S A Fuqua

Publications and source records attributed to S A Fuqua.

At least 91 records · Page 5Linked to original sources
PubMed1992

The importance of normal and abnormal oestrogen receptor in breast cancer.

We have used the screening techniques of CMC and SSCP of selected PCR fragments, and also gel retardation assays, to discover a number of ER variants in clinical breast cancer tissues. We have found base pair insertions, transitions, and deletions, and deletions of exons 3, 5 and 7. Using a yeast transactivation assay, we have discovered receptors with outlaw function consisting of both dominant positive receptors that were transcriptionally active in the absence of oestrogen and dominant negative receptors that were transcriptionally inactive themselves but prevented normal ER function. Future efforts should focus in particular on such dominant positive and dominant negative variants. With regard to positive variants, we should like to know whether they stimulate tumour growth and, if so, whether they can be turned off. With regard to dominant negative variants, we should like to determine whether they can inhibit tumour growth and, if so, whether they can be turned on.

Base Sequence↗
PubMed1991

Progesterone receptor gene restriction fragment length polymorphisms in human breast tumors.

We examined the progesterone receptor (PgR) gene in tissue from both primary human breast tumors and normal placentas, detecting restriction fragment length polymorphisms (RFLPs) with the restriction endonucleases Pst I/Sst I and HindIII. There was a general agreement of the Pst I and Sst I polymorphisms in any individual tumor, suggesting that they define two alleles in the human PgR locus, one being characterized by a deletion of about 300 base pairs with respect to the other. Both primary human breast tumor specimens (n = 36) and human term placentas (n = 48) displayed similar allele frequencies and typical mendelian distribution of these Pst I/Sst I alleles. The previously reported HindIII PgR RFLP was also investigated in 132 breast tumors. The HindIII PgR gene RFLP did not display typical mendelian distribution in the breast tumors; the factors affecting the HindIII allele frequencies are presently unknown. Neither the HindIII RFLP nor the deletion defined by Pst I and Sst I correlated with PgR expression as determined by a ligand-binding assay, suggesting that neither is related to the heterogeneity of PgR expression seen in breast tumors.

Blotting, Southern↗
PubMed1991

Variant human breast tumor estrogen receptor with constitutive transcriptional activity.

Since progesterone receptor (PgR) is normally induced by estrogen, breast cancer lacking estrogen receptor (ER) would also be expected to lack PgR. However, a small percentage of breast cancers are ER- yet PgR+. These tumors might possess an ER which is defective in estrogen binding but is still functional in stimulating estrogen-responsive genes such as PgR. We have now detected such a variant, lacking exon 5 of the hormone-binding domain, using complementary DNA amplified by the polymerase chain reaction. This variant was the predominate ER RNA expressed in three ER-/PgR+ tumors. Furthermore, the variant ER constitutively activates transcription of a normally estrogen-dependent gene construct in yeast cells. The variant ER could explain the expression of PgR in certain tumors and have therapeutic implications.

Base Sequence↗
PubMed1991

Association of INT2/HST1 coamplification in primary breast cancer with hormone-dependent phenotype and poor prognosis.

The human proto-oncogene INT2 (homologous to the mouse INT2 gene, implicated in proviral induced mammary carcinoma) has been mapped to chromosome 11q13 and found to share band localisation with, among others, the HST1 proto-oncogene. Both genes are members of the fibroblast growth factor family. In the present study, coamplification (2-15 copies) of the INT2/HST1 genes was found in 27 (9%) of 311 invasive human breast carcinomas using slot blot and Southern blot analyses. Amplification was not correlated to tumour size, axillary lymph node status or stage of disease, neither to patient age nor menopausal status. However, 26 (96%) of the 27 amplified tumours were, often strongly, Oestrogen receptor positive compared to 65% of the unamplified cases (P = 0.001). These findings are in sharp contrast to the strong correlations of HER-2/neu proto-oncogene amplification with advanced stage and steroid receptor negativity, previously observed in the same series of tumours. Patients with INT2/HST1 amplified breast cancer had a significantly shorter disease-free survival compared to those with unamplified genes (P = 0.015, median follow up 45 months). This correlation was confined to node-negative patients and persisted in multivariate analysis. No significant correlation to survival from breast cancer was found. It is concluded that amplification of the 11q13 region in breast cancer occurs in a particular subset of aggressive tumours, quite different from that identified by HER-2/neu amplification. It still remains to be shown that the selection for amplified genes at 11q13 is due to the activity of INT2, HST1 or yet another, still unidentified, neighbouring gene. However, the results are potentially of clinical value in separating a group of node-negative breast cancer for more intense treatment.

Adult↗
PubMed1991

Estrogen receptor variants in clinical breast cancer.

We have used the screening techniques of chemical mismatch cleavage, single stranded conformational polymorphism, and gel retardation to discover a number of estrogen receptor RNA variants in clinical breast cancer tissues. We have found basepair insertions, transitions, and deletions as well as alternative splicing, yielding deletions of exon 3, 5, or 7. Using a yeast transactivation assay we have discovered receptors with outlaw function, including both a dominant-positive receptor, which is transcriptionally active in the absence of estrogen, and a dominant-negative receptor, which is itself transcriptionally inactive, but prevents the action of normal estrogen receptor. These variants could have clinical significance, helping to explain breast tumor behavior and patient outcome.

Amino Acid Sequence↗
PubMed1990

HER-2/neu amplification predicts poor survival in node-positive breast cancer.

HER-2/neu protooncogene amplification and protein expression were analyzed with slot blot and Western blot techniques, respectively, in more than 300 invasive primary breast tumors of all stages. Amplification (2- greater than 30 copies) was found in 17% of these tumors and high expression was seen in 19%. There was a striking coincidence between gene amplification and high expression. Tumors associated with many involved axillary lymph nodes or with Stage IV disease were more often HER-2/neu amplified or overexpressed. Furthermore, gene alteration was strongly correlated with the absence of steroid receptors and with larger tumor size. High expression without gene amplification was seen in a minor subset of tumors of less aggressive character. Neither amplification nor overexpression was correlated with disease outcome for patients with negative axillary lymph nodes. For node-positive patients, however, HER-2/neu amplification was a significant predictor of early relapse and death (median follow-up = 45 months), and a similar trend, although not significant, existed for high gene expression. Multivariate analyses indicated that HER-2/neu alterations were not independent predictors of patient outcome.

Amino Acid Sequence↗
PubMed1990

Estrogen receptor gene methylation in human breast tumors.

DNA methylation is known to be involved in eukaryotic gene control; it may thus exert effects during development and tumorigenesis. We have examined the methylation status of the estrogen receptor (ER) gene in different human tissues. The ER gene was found to be methylated in placental tissues, but normal breast tissues exhibited a different methylation pattern. In addition, specific sites in the hormone-binding domain of the ER gene were observed to be differently methylated in different human breast tumor specimens. We did not detect, however, any association between the ER status of a tumor and ER gene methylation at these sites. Interestingly, a difference in the methylation status between normal and adjacent breast tumor tissues was observed. Thus, DNA methylation may be considered an additional molecular measure of the genetic heterogeneity in breast cancer.

Blotting, Southern↗
PubMed1990

Sensitive detection of estrogen receptor RNA by polymerase chain reaction assay.

We developed a simplified polymerase chain reaction (PCR) technique sensitive enough to detect estrogen receptor (ER) mRNA in breast tumor specimens from 1 microgram of total RNA. We simultaneously amplified a control gene such as beta-actin as a baseline to semiquantitate ER expression. In a preliminary test of this method on a small series of breast tumors, ER message was found as expected in a number of tumors found to be ER positive by ligand binding assay, but also ER negative in one tumor assayed. The ER in this last tumor contained a single base change in the hormone binding region, compared with the MCF-7 breast tumor cell line ER. Therefore, this PCR technique may be useful in the detection and cloning of rare ER transcripts from breast tumor biopsy specimens.

Actins↗
PubMed1990

Isolation and characterization of an adriamycin-resistant breast tumor cell line.

An adriamycin-resistant human breast tumor cell line MDA-A1R was generated by step-wise selection in increasing concentrations of drug from the parent cell line MDA-MB-231. MDA-A1R cells grow as loosely attached cell aggregates with a doubling time of 28-32 h; the MDA-MB-231 parent cell line grows as a standard monolayer culture with a 20-h doubling time. The MDA-A1R cell line is highly resistant to adriamycin compared to the parent cell line, and is cross-resistant to velban and colchicine suggestive of a multidrug resistance (MDR) phenotype. MDA-A1R cells exhibit reduced net adriamycin content as compared to the parent cell line. The MDR-associated P-glycoprotein gene is amplified approximately 10- to 30-fold in MDA-A1R cells. P-glycoprotein sequences are overexpressed in the resistant cells and are stable for up to 13 wk after drug removal. Moreover, MDA-A1R cells show the presence of very high levels of P-glycoprotein. MDA-A1R is thus an in vitro model system to study the mechanism of MDR in human breast cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts.

A simplified polymerase chain reaction (PCR) technique for the detection, semiquantitation and cloning of low-abundance RNAs is described. This assay involves first-strand cDNA synthesis by reverse transcription of mRNA with specific oligonucleotide primers, followed by second-strand synthesis and PCR amplification, in the same tube with only the addition of TaqI DNA polymerase. The assay is sufficiently sensitive to detect target RNA from as little as 1 ng of total RNA. The beta-actin transcripts may also be simultaneously reverse transcribed, amplified and used as an internal standard to determine relative expression of specific RNAs. Using this simple technique, expression of the multidrug resistance (mdr 1) gene can easily be detected in human breast tumors. The technique is also applicable for the cloning of rare transcripts.

Actins↗
PubMed1989

Induction of the estrogen-regulated "24K" protein by heat shock.

We have previously demonstrated an estrogen-regulated protein, termed 24K, in human breast cancer cell lines and human tumor biopsies. The presence of the protein correlates well with the presence of steroid hormone receptors. We have cloned a partial complementary DNA to 24K and show that the nucleotide and deduced amino acid sequence contains a striking homology to the low molecular weight heat shock proteins of Drosophila and the mammalian alpha-crystallins. Our complementary DNA is identical to that reported for the 3' region of human heat shock protein 27/28 with the mRNAs for the gene significantly induced by both heat shock and estradiol treatment in MCF-7 breast cancer cells. A variant polymorphism of the gene was detected in two estrogen-unresponsive cell lines, but not in other human breast cancer cell lines or human placenta; the polymorphism did not affect heat shock induction of the gene. The heat shock protein 27/28 model system in MCF-7 cells will afford a valuable tool to analyze the molecular events underlying the hormonal regulation of gene expression; heat shock may offer an approach to manipulate the hormonal regulation of the heat shock protein 27/28 gene.

Base Sequence↗
PubMed1989

Estrogen receptor expression in human breast cancer associated with an estrogen receptor gene restriction fragment length polymorphism.

Estrogen receptor (ER) content is a well-known predictor of clinical outcome in human breast cancer. The recent cloning of a human ER complementary DNA has made possible the characterization of the ER gene on a molecular level. We have examined in human breast cancers a single, two-allele restriction fragment length polymorphism using the restriction enzyme PvuII. Initial studies in human breast cancer cell lines suggested a possible association between the absence of one allele and the absence of ER expression; subsequent analysis of allele distribution and frequency in 188 primary human breast tumor biopsies did indeed show a significant but not complete correlation between the absence of one allele and the failure to express ER. Preliminary data suggest that this restriction fragment length polymorphism is located within gene sequences coding for the putative DNA or hormone-binding domains of the ER.

Alleles↗
PubMed1989

Chromosomal assignments of human 27-kDa heat shock protein gene family.

The 27-kDa human heat shock protein (hsp27) is dually regulated by both estrogen and heat shock treatment. Its function is obscure, but the high degree of homology to the lens alpha-crystallins and to the small heat shock proteins of Drosophila suggests that it may serve a structural function. There are at least three related human hsp27 sequences. To provide possible insight into the dual regulation of hsp27, we have determined the chromosomal location of all these and found them to be on separate chromosomes: 3, 9, and X.

Animals↗
PubMed1989

Electrophoretic analysis of 248 clinical breast cancer specimens for P-glycoprotein overexpression or gene amplification.

Multiple drug resistance (MDR), consisting of acquired cross resistance to anthracyclines, vinca alkyloids, and other antineoplastic antibiotics, has been described in a variety of cell lines. This MDR phenotype is associated with overexpression and sometimes amplification of a gene coding for a 170 kDa glycoprotein, termed P-glycoprotein. To understand the role of this mechanism in clinical breast cancer, 248 breast cancer specimens representing both untreated primary and refractory relapsing disease were probed for evidence of P-glycoprotein gene amplification or overexpression using Southern, Northern, or Western blot techniques. In no case was an increase in P-glycoprotein gene copy number or expression detected. Though these findings do not necessarily rule out a role for P-glycoprotein in mediating drug resistance in breast cancer, electrophoretic analysis of clinical specimens is unlikely to provide useful predictive information. More sensitive assays must be developed to overcome the difficulties inherent in analyzing heterogenous tissue samples.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Insulin-like growth factor-II (IGF-II): a potential autocrine/paracrine growth factor for human breast cancer acting via the IGF-I receptor.

Insulin-like growth factor-II (IGF-II) is a potent mitogen for several types of cultured cells and tissues. We have studied the interaction of IGF-II with a panel of cultured human breast cancer cell lines, examining the possibility that these cells synthesize and secrete IGF-II activity which could have autocrine/paracrine functions. Synthetic IGF-II was mitogenic in five of seven cell lines tested, including the estrogen receptor-positive lines MCF-7L, ZR75-1, and T47D and the estrogen receptor (ER)-negative lines Hs578T and MDA-231. IGF-II was slightly less potent than IGF-I in stimulating DNA synthesis in MCF-71 cells, an effect that paralleled its ability to compete for [125I]IGF-I binding in these cells. Affinity labeling studies revealed that IGF-II could also compete for binding to the 130,000 mol wt alpha-subunit of the IGF-I receptor. A monoclonal antibody to the IGF-I receptor inhibited the mitogenic effects of IGF-II in MCF-7L and MDA-231 cells, suggesting that this receptor mediates the growth effects of IGF-II in these breast cancer cells. Using a RIA and a RRA, IGF-II-like activity was detected in conditioned medium extracts processed to remove IGF-binding proteins from several breast cancer cell lines, with the highest levels found in conditioned medium from MCF-7L and T47D cell lines. IGF-II mRNA transcripts in MCF-7L and T47D cells were identified by Northern blot analysis and were confirmed by RNase protection assay. IGF-II mRNA was increased by estrogen in MCF-7L cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1988

A cDNA for the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells.

We have previously demonstrated an estradiol-regulated 24 kDa (24K) protein in human breast cancer tissue culture cells and human tumor biopsies. The presence of 24K correlates well with the presence of steroid hormone receptors. In order to further study the hormonal regulation of the 24K protein and gene, we have isolated cDNA clones corresponding to the 24K mRNA. Poly(A)+ RNA isolated from the MCF-7 human breast cancer cell line was translated in a cell-free translation system containing [35S]-methionine. The translation products were immunoprecipitated with a 24K monoclonal antibody, and the in vitro synthesis of 24K protein was confirmed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. The same poly(A)+ RNA was used to construct an oligo(dT)-primed cDNA library in the lambda gt11 expression vector system. The library was screened with a highly specific polyclonal antibody raised against 24K protein purified by immunoaffinity chromatography. Four recombinant clones reacting with the antibody by virtue of antigen expression were isolated and three were used in hybridization-selected translation. Three clones were able to hybridize specifically to a messenger RNA (mRNA) that yielded a Mr 24,000 protein when translated in vitro and analyzed by SDS/polyacrylamide gel electrophoresis. This protein was also immunoprecipitable by the 24K monoclonal antibody. MCF-7 mRNA size fractionated by formaldehyde-agarose gel electrophoresis was transferred to nitrocellulose paper and hybridized to a nick-translated 24K cDNA clone. A single band of hybridization corresponding to a mRNA size of approximately 0.9-1.0 kilobase (kb) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗