Yeast Screens Identify the RNA Polymerase II CTD and SPT5 as Relevant Targets of BRCA1 Interaction

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression

\u3cem\u3ec-erbB-2\u3c/em\u3e 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

\u3cem\u3ec-erbB-2\u3c/em\u3e 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

Sensitive and Specific Detection of the 4B5 Antigen in Bronchial Lavage Specimens from Patients with Primary Bronchogenic Carcinoma

The monoclonal antibody 4B5 binds to a mucin-like antigen elaborated by respiratory epithelium of patients with non-small cell bronchogenic carcinoma. Several immunoassay formats were used to determine the presence of the antigen in lavage specimens. A qualitative immuno-drop binding assay showed immunoreactivity in 37 (64%) of 58 specimens from patients with non-small cell lung cancer. In contrast, only 11 (12%) of 93 specimens from patients with either metastatic carcinoma or benign pulmonary diseases exhibited 4B5 immunoreactivity. A quantitative radioimmunoassay using standardized amounts of mucin exhibited similar sensitivity and specificity. Positive immunoreactivity was associated significantly with tobacco use and the cytopathologic diagnoses of squamous metaplasia, atypia, or dysplasia. Conversely, no significant association was found between 4B5 immunoreactivity and age, gender, race, benign cyto-logic findings, frankly malignant cytologic findings, or stage of disease. The expression of 4B5 antigen in bronchial secretions from patients with bronchogenic carcinoma deserves additional evaluation as a potential marker of pulmonary carcinogenesis

Sensitive and Specific Detection of the 4B5 Antigen in Bronchial Lavage Specimens from Patients with Primary Bronchogenic Carcinoma

The monoclonal antibody 4B5 binds to a mucin-like antigen elaborated by respiratory epithelium of patients with non-small cell bronchogenic carcinoma. Several immunoassay formats were used to determine the presence of the antigen in lavage specimens. A qualitative immuno-drop binding assay showed immunoreactivity in 37 (64%) of 58 specimens from patients with non-small cell lung cancer. In contrast, only 11 (12%) of 93 specimens from patients with either metastatic carcinoma or benign pulmonary diseases exhibited 4B5 immunoreactivity. A quantitative radioimmunoassay using standardized amounts of mucin exhibited similar sensitivity and specificity. Positive immunoreactivity was associated significantly with tobacco use and the cytopathologic diagnoses of squamous metaplasia, atypia, or dysplasia. Conversely, no significant association was found between 4B5 immunoreactivity and age, gender, race, benign cyto-logic findings, frankly malignant cytologic findings, or stage of disease. The expression of 4B5 antigen in bronchial secretions from patients with bronchogenic carcinoma deserves additional evaluation as a potential marker of pulmonary carcinogenesis