Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors.

There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

Nucleolar localization of BRCA1 protein in human breast cancer.

Both a rabbit polyclonal BRCA1 antibody, K-18, and a mouse monoclonal BRCA1 antibody, AP 16, produced nucleolar epithelial cell staining on frozen tissue sections of human infiltrating mammary carcinomas. There was much less BRCA1 antibody staining in normal tissues; however, 2 intraductal tumors and a papilloma, found in proximity to the carcinomas showed considerable nucleolar immunoreactivity. MCF-7 cells fixed in methanol and immunostained with the same two antibodies also revealed nucleolar staining, however, after 4% paraformaldehyde fixation for three minutes, there were many fewer nuclei stained. Antigen retrieval methods on formalin-fixed, paraffin-embedded specimens produced tumor cell cytoplasmic staining with AP 16 and nuclear staining in both tumor and normal epithelial cells with another BRCA1 monoclonal antibody, SG 11.

Adeno-associated virus gene transfer into renal cells: potential for in vivo gene delivery.

The human parvovirus adeno-associated virus (AAV), type 2, has a number of features that make it an attractive choice as a vector for gene delivery to the kidney. AAV vectors permit long-term gene expression in vivo by integration into the host genome, have potential for site-specific integration on chromosome 19, do not express viral genes or generate a cellular immune response, and demonstrate enhancement of gene expression by chemotherapeutic agents that are approved for use in vivo. These properties confer advantages to AAV over other viral and nonviral methods for gene transfer. Preliminary experiments in our laboratory suggest that AAV is able to transfer genes to both renal cells in culture and the kidney in vivo. Thus, AAV has the potential to be an important gene transfer vector for the kidney in vivo.

Immunohistologic c-myc protein in benign breast disease and cancer.

We have studied the histopathology and differential distribution of the c-myc protein (Myc) in human breast tissues including 17 cases of infiltrating mammary carcinoma, 4 cases of fibroadenoma, 5 cases with fibrocystic changes, and 1 case of reduction mammoplasty (as a control). Using a sensitive immunohistochemical method on frozen tissue sections, both a rabbit polyclonal anti-c-myc antibody and a mouse monoclonal anti-c-myc antibody, H51C116, produced high levels of Myc staining in the nuclei of epithelial cells of infiltrating mammary carcinomas (30-90% of cells stained). In contrast, the nuclei of epithelial cells of fibroadenomas, and breast tissues with fibrocystic changes stained infrequently. We studied benign tissue surrounding the tumors in four cases; three were essentially negative, and one showed nuclear epithelial cell staining throughout the lobules. Sixteen of the tumors were examined in parallel, using formalin-fixed, paraffin-embedded samples. Immunohistological procedures for Myc produced uniform, intense epithelial cell cytoplasmic staining (8 cases); light epithelial cell cytoplasmic staining (5 cases) or were unstained (3 cases). We argue that the differences between frozen and paraffin sections are incompatible with the notion of simple displacement of nuclear Myc to the cytoplasm during fixation. Elevated levels of nuclear Myc in tumor cells and subsets of benign tissue are consistent with a role for Myc in mammary cell proliferation and tumorigenesis.

C-myc protein distribution - mammary adenocarcinomas of mtv/myc transgenic mice.

Transgenic mice have been used to demonstrate that the myc transgene, fused to the murine mammary tumor virus LTR promoter, leads to development of mammary tumors. To study the role of the Myc protein in these tumors, a sensitive immunohistochemical method was used to compare the Myc protein expression in mammary tumors and normal mammary gland from two independent MTV/myc transgenic lines. The highest levels of staining for Myc were found in the epithelial cell nuclei of mammary tumors and foci of mammary hyperplasia. Normal transgenic epithelium had only scattered foci of low intensity staining nuclei. The nuclei of transgenic mesenchyme and of non-transgenic mammary glands of control FVB mice did not contain detectable antigen. This work supports the hypothesis that the Myc protein can play a role in mammary tumorigenesis and suggests a correlation between high levels of Myc and the development of histopathology.

Distribution of c-myc oncoprotein in healthy and atherosclerotic human carotid arteries.

PURPOSE: Smooth muscle cell (SMC) proliferation is a central event in the development of arteriosclerotic plaque. Regulation of this proliferative process is controlled in part by the action of specific peptide growth factors that may influence early cell-cycle regulatory gene expression. Such "early" response genes include the protooncogene c-myc, which has been implicated in the induction of cell proliferation and differentiation. We compared the distribution of the c-myc protooncogene product in healthy and atherosclerotic human carotid arteries to determine its cellular and tissue localization. METHODS: Samples of six carotid artery plaques from six patients were rapidly frozen in liquid nitrogen at the time of carotid endarterectomy. Three nondiseased human carotid arteries obtained at organ harvest from brain-dead organ donors were similarly prepared. Frozen sections were labeled with a polyclonal rabbit anti-c-myc antibody that recognizes the 64 kd c-myc human protein. The percentages of cells positive for c-myc (c-myc index) and the intensity of antibody labeling were determined. RESULTS: Normal human carotid artery demonstrated minimal, isolated cell staining, with single scattered grains of immunocytochemical staining product seen in SMC nuclei. The myc index was 14.7% +/- 3.5% positive cells. In comparison, SMCs from carotid plaque showed a significant predominance of c-myc immunoreactive cells (89.8% +/- 4%; p < 0.001). The intensity of c-myc staining was greater in plaque SMCs, with many of the cells demonstrating confluence of immunocytochemical precipitate throughout 50% of SMC nuclei. CONCLUSIONS: Although the exact role of enhanced expression of the c-myc protooncogene in atherosclerosis is unclear, a cooperative influence of abnormal early cell-cycle gene expression and humoral factors may initiate the atherogenic process. The c-myc gene and other protooncogenes are early molecular markers of cell-cycle activity, which may be important in the development of atherosclerosis and occlusive vascular disease.

c-myc protein distribution. Neoplastic tissues of the human colon.

There is an extensive literature documenting the increased or deregulated expression of the c-myc oncogene in human malignancies. The authors have recently devised a sensitive immunocytochemical method for studying the tissue localization of c-myc protein in tissue sections of human colon. We have compared nuclear c-myc staining using a polyclonal rabbit anti-c-myc antibody and a mouse monoclonal myc antibody NCM II 274. Microscopic observation of the tissue specific pattern of c-myc protein distribution shows that nuclear staining intensity varies in normal and neoplastic crypt cell nuclei in parallel with morphologic criteria of neoplasia. These studies yield further information on the usefulness of c-myc protein as a prognostic indicator.

Distribution of the c-myc oncoprotein in normal and neoplastic tissues of the rat colon.

A rat model of 5-azoxymethane induced colon cancer was studied in order to correlate histopathological changes and the differential distribution of the c-myc protein. Weanling Fisher 344 rats were injected with three, one week apart, subcutaneous injections of 5-azoxymethane (AOM) (15 mg kg-1) and the animals were divided into low and high fat diet groups. Nine colon tumors, of varying degrees of malignancy, that developed in the AOM-treated rats, and sections of normal colonic mucosa were examined. A rabbit polyclonal anti-c-myc antibody produced nuclear staining at 1:100 dilution in cryostat frozen sections of the normal rat colonic mucosa and the colon tumors when prepared with a Cryostat Frozen Sectioning Aid (CFSA). The tissue localization of the c-myc antibody staining revealed: (1) in normal mucosa, nuclei of the basal portion of the mucosa; (2) in adenomatous polyps, nuclei at all levels of the mucosa; and (3) in a carcinoma in situ, intense staining of glandular epithelial cell nuclei at all levels within the tumor. This procedure may provide a sensitive method for detecting abnormal cells in the colonic epithelium that have an altered proliferative capacity.

gamma-Glutamyl cyclotransferase: a new genetic polymorphism in the mouse (Mus musculus) linked to Lyt-2.

Electrophoretically variant forms of gamma-glutamyl cyclotransferase have been identified in red cells of inbred mouse strains. Each inbred strain exhibited a major band of activity and a minor band that migrated more anodally. The polymorphism affects the migration of both the major and minor bands in a similar way. F1 hybrids between strains with fast forms (A/J) and strains with the slow forms (C57BL/6J) exhibited a four-banded pattern consistent with co-dominant inheritance. The patterns observed in backcross and F2 mice were consistent with the segregation of a pair of autosomal co-dominant alleles. Recombinant inbred strains and a congenic strain were used to show that the locus controlling gamma-glutamyl cyclotransferase (Ggc) is linked to Lyt-2, a lymphocyte alloantigen locus on chromosome 6, with an estimated map distance of 5.0 +/- 2.5 centimorgans.

Linkage of gene for C2 deficiency and the major histocompatibility complex MHC in man. Family study of a further case.

Close linkage between HL-A and C2 deficiency was first reported by FU and co-workers in 1974. We present here a pedigree of a 31-year-old C2-deficient individual with clinical manifestations of Hodgkins disease. The following markers were tested: C2 levels, factor B polymorphism, blood groups, and enzyme typing. In addition to close linkage between HL-A and C2 deficiency, both parents were heterozygous for Bf (HL-A linked, electrophoretic variation of B). The two HL-A haplotypes closely linked to C2 deficiency are different: 2, W18 and W24, W18. They share, however, the SD2 antigen W18 and the LD type 7a.

C3, GBG, orosomucoid and haptoglobin polymorphisms. Improved staining methods.

C3, GBG, and orosomucoid polymorphisms were electrophoresed in a high-voltage agarose method which permitted the typing of 15-20 samples. The increased sensitivity of the dye Coommassie blue was used to stain the protein, and yielded higher resolution than amido black. The typing of haptoglobin samples, was facilitated by devising a method which utilizes the peroxidase activity of the haptoglobin-hemoglobin complex with 90-tolidine and hydrogen peroxide as substrates and 4-chloro-1-naphthol as coupler.