Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation.

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, nonviral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable the development of enhanced nonviral gene transfer in salivary glands and other tissues. One potential barrier is the degradation of plasmid DNA by endonucleases. To test this hypothesis, we coadministered two endonuclease inhibitors ((zinc and aurintricarboxylic acid (ATA)) with plasmid DNA, containing the secreted alkaline phosphatase gene (SEAP), to the submandibular glands of rats. The effect of zinc and ATA on SEAP expression, tissue accumulation of plasmid DNA, and plasmid DNA stability was then characterized. We observed that mixtures containing zinc/DNA, ATA/DNA, and zinc/ATA/DNA significantly enhanced both systemic transgene expression and the amount of plasmid DNA associated with treated tissues. The relative endonuclease inhibitory activity of zinc, ATA, and zinc/ATA correlated with the observed effects on transfection efficacy. The use of zinc/ATA enhanced the efficacy of salivary gland transfection by at least 1000-fold versus DNA alone. Importantly, this improved performance resulted in robust systemic secretion of an exogenous protein (SEAP), thus demonstrating the potential this nonviral gene transfer technology has as a method to treat systemic protein deficiencies.

Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease.

The HER-2/neu proto-oncogene (also known as c-erb B-2) is homologous with, but distinct from, the epidermal growth factor receptor. Amplification of this gene in node-positive breast cancers has been shown to correlate with both earlier relapse and shorter overall survival. In node-negative breast cancer patients, the subgroup for which accurate prognostic data could make a significant contribution to treatment decisions, the prognostic utility of HER-2/neu amplification and/or overexpression has been controversial. The purpose of this report is to address the issues surrounding this controversy and to evaluate the prognostic utility of overexpression in a carefully followed group of patients using appropriately characterized reagents and methods. In this report we present data from a study of HER-2/neu expression designed specifically to test whether or not overexpression is associated with an increased risk of recurrence in node-negative breast cancers. From a cohort of 704 women with node-negative breast cancer who experienced recurrent disease (relapsed cases) 105 were matched with 105 women with no recurrence (disease-free controls) after the equivalent follow-up period. Immunohistochemistry was used to assess HER-2/neu expression in archival tissue blocks from both relapsed cases and their matched disease-free controls. Importantly, a series of molecularly characterized breast cancer specimens were used to confirm that the antibody used was of sufficient sensitivity and specificity to identify those cancers overexpressing the HER-2/neu protein in this formalin-fixed, paraffin-embedded tissue cohort. In addition, a quantitative approach was developed to more accurately assess the amount of HER-2/neu protein identified by immunostaining tumor tissue. This was done using a purified HER-2/neu protein synthesized in a bacterial expression vector and protein lysates derived from a series of cell lines, engineered to express a defined range of HER-2/neu oncoprotein levels. By using cells with defined expression levels as calibration material, computerized image analysis of immunohistochemical staining could be used to determine the amount of oncoprotein product in these cell lines as well as in human breast cancer specimens. Quantitation of the amount of HER-2/neu protein product determined by computerized image analysis of immunohistochemical assays correlated very closely with quantitative analysis of a series of molecularly characterized breast cancer cell lines and breast cancer tissue specimens.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.

Carcinoma of the breast and ovary account for one-third of all cancers occurring in women and together are responsible for approximately one-quarter of cancer-related deaths in females. The HER-2/neu proto-oncogene is amplified in 25 to 30 percent of human primary breast cancers and this alteration is associated with disease behavior. In this report, several similarities were found in the biology of HER-2/neu in breast and ovarian cancer, including a similar incidence of amplification, a direct correlation between amplification and over-expression, evidence of tumors in which overexpression occurs without amplification, and the association between gene alteration and clinical outcome. A comprehensive study of the gene and its products (RNA and protein) was simultaneously performed on a large number of both tumor types. This analysis identified several potential shortcomings of the various methods used to evaluate HER-2/neu in these diseases (Southern, Northern, and Western blots, and immunohistochemistry) and provided information regarding considerations that should be addressed when studying a gene or gene product in human tissue. The data presented further support the concept that the HER-2/neu gene may be involved in the pathogenesis of some human cancers.

Progesterone receptor distribution in the human endometrium. Analysis using monoclonal antibodies to the human progesterone receptor.

Two monoclonal antibodies to the human progesterone receptor (PR), JZB39 and KD68, were used in determining the immunohistochemical distribution of PR in the human endometrium throughout the menstrual cycle and after menopause. These antibodies recognized PR, as demonstrated by a downfield shift in the radiolabeled progestin binding peak when KD68 or JZB39 was added to high salt sucrose density gradients. The specificity of both antibodies for PR was confirmed with Western immunoblots and competition studies performed with purified receptor. Progesterone receptor was identified with these antibodies and the peroxidase-antiperoxidase technique in the nuclei of epithelial cells, stromal cells, and myometrial smooth muscle cells. The receptor content of endometrial epithelium and stroma varied with the menstrual cycle. The variation was most marked in the epithelium, which demonstrated very strong PR immunostaining during the proliferative phase and postovulation Days 1-3 of the early secretory phase, but PR immunostaining decreased sharply at postovulation Day 4 and remained relatively weak or absent during the mid and late secretory phase. In contrast, stromal cell nuclei were moderately to strongly immunostained even during the secretory phase. Progesterone receptor was not localized in vascular smooth muscle cells or endothelial cells. Specific cytoplasmic staining for PR was not identified in any of these cases, even prior to ovulation, when circulating levels of progesterone are low, indicating that both the steroid-occupied and -unoccupied forms of human progesterone receptor, like rabbit and guinea pig PR, and estrogen receptor, is a nuclear protein.