Gudrun Huper

BRCA1 expression is not directly responsive to estrogen

Expression of the breast cancer susceptibility gene, BRCA1, is induced by 17-β estradiol (E2) in estrogen receptor containing breast cancer cell lines. Our previous studies have shown that BRCA1 transcription is also regulated with the cell cycle, reaching maximal levels just before the onset of DNA synthesis. In this study, we have examined whether the estrogen induction of BRCA1 is direct or is a result of the mitogenic activity of the hormone. Four lines of evidence lead us to conclude that E2 induces BRCA1 primarily through an increase in DNA synthesis: (1) The kinetics and magnitude of induction are different from the directly E2 inducible gene, pS2; (2) Induction of BRCA1, but not pS2, is blocked by cycloheximide indicating that de novo protein synthesis is required; (3) Other hormonal and growth factor treatments that induce DNA synthesis have a similar effect, including IGF-1, EGF and DNA synthetic flares induced by tamoxifen and retinoic acid; (4) BRCA1 genomic fragments near the 5′ end of the gene containing putative estrogen response elements fail to respond to E2 when transfected into breast cancer cell lines. The most consistent explanation for these findings and other published studies is that BRCA1 transcription is induced as a result of the mitogenic activity of E2 in estrogen receptor positive cells.

Polypeptides of avian RNA tumor viruses. IV. Components of the viral envelope

Abstract By treatment of avian myeloblastosis virus (AMV) with NP 40, it was possible to quantitatively isolate a rosettelike structure whose subunits are related to the surface projections of the virus. Detailed analysis of this material resulted in the following findings: (i) The rosettes represented about 7% of the protein and 60% of the carbohydrate content of the virus. (ii) SDS-polyacrylamide gel electrophoresis indicated the presence of two glycoproteins having molecular weights of 37,000 (GI) and 115,000 (GII). (iii) GII could be isolated without treatment with SDS and structurally resembled the knob portion of the rosettes. (iv) Both GI and GII specifically absorbed neutralizing sera and precipitated with these in immunodiffusion analysis. (v) In addition to type-specific antigenicity, two distinct antigenic determinants were demonstrated in the rosettes utilizing nonneutralizing rabbit sera. One of these resided in GI and the other in GII. These antigens were shown to be distinct from the major complement-fixing antigens of the virus. These results confirm the belief that the surface projections of avian RNA tumor viruses represent the type-specific antigens.

Analysis of methylation-sensitive transcriptome identifies GADD45a as a frequently methylated gene in breast cancer

Treatment of the breast cancer cell line, MDAMB468 with the DNA methylation inhibitor, 5-azacytidine (5-AzaC) results in growth arrest, whereas the growth of the normal breast epithelial line DU99 (telomerase immortalized) is relatively unaffected. Comparing gene expression profiles of these two lines after 5-AzaC treatment, we identified 36 genes that had relatively low basal levels in MDAMB468 cells compared to the DU99 line and were induced in the cancer cell line but not in the normal breast epithelial line. Of these genes, 33 have associated CpG islands greater than 300 bp in length but only three have been previously described as targets for aberrant methylation in human cancer. Northern blotting for five of these genes (α-Catenin, DTR, FYN, GADD45a, and Zyxin) verified the array results. Further analysis of one of these genes, GADD45a, showed that 5-AzaC induced expression in five additional breast cancer cell lines with little or no induction in three additional lines derived from normal breast epithelial cells. The CpG island associated with GADD45a was analysed by bisulfite sequencing, sampling over 100 CpG dinucleotides. We found that four CpGs, located approximately 700 bp upstream of the transcriptional start site are methylated in the majority of breast cancer cell lines and primary tumors but not in DNA from normal breast epithelia or matched lymphocytes from cancer patients. Therefore, this simple method of dynamic transcriptional profiling yielded a series of novel methylation-sensitive genes in breast cancer including the BRCA1 and p53 responsive gene, GADD45a.

Polypeptides of mammalian oncornaviruses: III. Localization of p15 and reactivity with natural antibody

Abstract Antiserum to p15 of Friend murine leukemia virus (FLV) reacted with intact MuLVs in radioimmunoprecipitation (RIP) assays. The antigen-antibody complexes formed by this reaction were isolated and shown to contain p15 after analysis by SDS polyacrylamide gel electrophoresis. Natural antibody in mice to MuLV reacted in a similar fashion with p15, but in addition, also with the virus glycoproteins (gp71, gp45). Biological studies indicated that gp71 rather than p15 is principally involved in the virus-neutralization reaction. These and other studies indicate that p15 is localized on the virus surface and is recognized by natural antibodies in mice to MuLV.

Polypeptides of avian RNA tumor viruses: V. Analysis of the virus core

Abstract Disruption of avian myeloblastosis virus (AMV) with NP 40 followed by a brief treatment with cold ether led to a quantitative isolation of structures resembling the virus core. Analysis of the cores indicated that they contained the high molecular weight virus RNA, a major polypeptide (28,000 daltons molecular weight) which represents a group-specific (gs) antigen, and RNA and DNA polymerizing activities. Treatment of the cores with various enzymes and examination in the electron microscope indicated that both lipid and protein were required for their integrity. When the core preparations were applied to chick embryo fibroblast cultures, virus particles containing gs antigen and high-molecular-weightRNA were found in the supernatants after two cell passages, indicating the presence of infectious material.

Polypeptides of mammalian oncornaviruses: II. Characterization of a murine leukemia virus polypeptide (p15) bearing interspecies reactivity☆

Polypeptide p15 from Friend leukemia virus was isolated by multiple gel filtration steps in guanidine hydrochloride. Because of its marked tendency to aggregate, renaturation of the protein was performed in the presence of 0.2% sodium deoxycholate. Serological analyses revealed cross reactivity with other mammalian C-type oncornaviruses.

Maintenance of DNA content and erbB-2 alterations in intraductal and invasive phases of mammary cancer

SummaryDuctal carcinomain situ (intraductal carcinoma) of the breast is a commonly recognized and curable clinical entity. Patients with intraductal carcinoma are at risk to develop invasive breast cancer presumably due to a transition from the noninvasive to the invasive phase of growth. Primary breast malignancies commonly display bothin situ and invasive phases of growth in the same tumor. In the current study, DNA content and alterations in the erbB-2 (HER-2/neu) oncogene product were examined simultaneously in both growth phases of primary breast cancers by image analysis. DNA content in the intraductal and invasive components of primary breast cancers were virtually identical (r = 0.979, p < 0.001). Quantitative image analysis was used to measure erbB-2 expression and categories of expression were related to copy number of the erbB-2 gene. Expression of erbB-2 was similar in both growth phases and implies identity of the erbB-2 genotype. The identity of DNA content suggests that the noninvasive and invasive phases within a single breast cancer are highly related. It is likely that erbB-2 gene number remains the same during progression from intraductal to invasive disease.

Immunological properties of avian oncornavirus polypeptides

Abstract The major polypeptides and glycoproteins of avian myeloblastosis virus and of the Prague strain of Rous sarcoma virus were isolated by gel filtration in guanidine hydrochloride (GuHCl). Hyperimmune sera prepared in rabbits against homogeneous preparations of each material were used to study the nature of the antigenic specificities on these molecules. The results indicated that (1) each component contained unique antigenic determinants; (2) each of four polypeptides (27,000, 19,000, 15,000, 12,000 daltons) contained group-specific (gs) reactivity; and (3) subgroup specific reactivity was found in the 19,000-dalton polypeptide.

c-erbB-2 Expression in Breast Cancer Detected by Immunoblotting and Immunohistochemistry

Evidence that the c-erbB-2 proto-oncogene is important in prognosis and oncogenesis in a number of human malignancies is increasing. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized to determine the amplification and protein expression of this proto-oncogene, respectively. These extraction techniques are often time consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Immunohistochemistry (IHC), on the other hand, is more often time and cost effective. In addition, IHC may offer enhanced sensitivity over extraction techniques because of the in situ nature of analysis. In data presented here, 71 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilution DNA hybridization and IHC, respectively. In 65 (92%) of 71 cases, high-level expression was associated with gene amplification, whereas moderate or low-level expression was associated with a normal diploid gene copy number. In five of the six discrepant cases, IHC predicted amplification which was not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. In 39 of the 71 total cases, Western immunoblotting was compared with IHC in the assessment of oncoprotein expression. Concordance was found in 33 (85%) of 39 cases. In four of the six discrepant cases, high levels of c-erbB-2 expression were demonstrated by IHC but not by immunoblotting. In these cases, intraductal disease and stroma-rich tumors again led to a relative paucity of neoplastic tissue within the specimens. We conclude that IHC offers a favorable alternative to either Southern analysis or Western immunoblotting in the assessment of c-erbB-2 gene copy number and expression levels of oncoprotein in human mammary carcinoma. Furthermore, IHC may prove advantageous to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

Evaluation of expression based markers for the detection of breast cancer cells.

SummaryIntroductionGenes that are expressed in a highly tissue- or disease-specific manner provide possible targets for therapeutics, early detection of cancer, and monitoring of disease burden during and after treatment. Further, genes of this type that code for secreted or shed proteins may allow for serum detection of the product facilitating our ability to specifically detect the cancer in all circumstances. To this end, we are working towards identification and characterization of such genes that are specifically expressed in breast epithelium. In the current study, we have measured the expression of two markers that emerged from a screen of the Incyte LifeSeq Database and were subsequently shown to be highly restricted to breast epithelium termed BU101 (also called Lipophilin B) and BS106 (small mucin-like protein). These two novel markers were compared with two other candidate markers, Mammaglobin and Cytokeratin 19 (CK19).MethodsUtilizing quantitative real-time PCR, we compared the expression of these four genes in a series of 95 primary breast cancers, 9 lymph nodes from breast cancer patients, 13 lymph nodes from non-cancer patients and 10 normal breast tissues.ResultsCytokeratin was shown to be highly sensitive in detecting all breast cancers, while BU101, BS106 and Mammaglobin were more restricted.ConclusionWhile no one of the these markers efficiently detects all breast cancers, a combination of two or more could achieve a very high sensitivity in assaying for circulating or occult breast cancer cells.