PubMed Health⌕ Search

Biomedical subjects

S F Dowdy

Publications and source records attributed to S F Dowdy.

13 recordsLinked to original sources
PubMed1992

Expression, purification, and functional characterization of adenovirus 5 and 12 E1A proteins produced in insect cells.

The 12 S and 13 S E1A cDNAs from both the Adenovirus (Ad) nononcogenic type 5 and the oncogenic type 12 were overexpressed in an insect cell/baculovirus system. Upon infection of Spodoptera frugiperda cells, the production of E1A proteins reached a level of about 15 micrograms/10(6) cells. The E1A proteins are highly soluble and apparently are processed authentically. They are readily recognized by various antibodies and display phosphorylation patterns similar to those of E1A proteins synthesized in mammalian cells. Single-step immunoaffinity chromatography was used to purify the Ad5 E1A proteins to near homogeneity under nondenaturing conditions. The Ad5 and Ad12 E1A proteins are able to form complexes with the retinoblastoma susceptibility gene product (Rb) and other cellular proteins. Interestingly, the presence of a cellular extract seems to be a prerequisite for association between highly purified E1A and Rb polypeptides.

Adenoviridae↗
PubMed1992

The retinoblastoma protein and the regulation of cell cycling.

Increasing attention has been focused on how the retinoblastoma (RB) protein regulates cell growth. Recent evidence indicates that it is a substrate for phosphorylation by cyclin-dependent kinase-cyclin complexes and suggests that this phosphorylation modulates the ability of this protein to regulate transit through the cell cycle, perhaps in its G1 phase.

Animals↗
PubMed1991

The isolation and characterization of a novel cDNA demonstrating an altered mRNA level in nontumorigenic Wilms' microcell hybrid cells.

Wilms' tumor, a pediatric nephroblastoma, has been associated with genetic alterations of the 11p13 and 11p15 regions. The introduction of a der(11) chromosome into the G401 Wilms' tumor cell line has been shown previously to revert the tumorigenic phenotype. A subtractive cDNA/RNA hybridization performed between the tumorigenic parent (G401) and a nontumorigenic microcell hybrid of G401 (110.1/G401.1) containing the der(11) chromosome resulted in the identification of a single novel cDNA clone, designated QM. The cDNA is 745 nucleotides in length and encodes a predicted hydrophilic 25 kd basic protein, primarily consisting of alpha helices. The QM transcript is expressed in a wide variety of embryonic and adult tissues and demonstrates a down regulation of expression in adult kidney and heart. QM is also a member of a multigene family members of which map to chromosomes 6 and 14. The QM mRNA level is modulated between the tumorigenic and nontumorigenic cell lines and therefore may be involved in the maintenance of the nontumorigenic phenotype.

Amino Acid Sequence↗
PubMed1991

Suppression of tumorigenicity in Wilms tumor by the p15.5-p14 region of chromosome 11.

Wilms tumor has been associated with genomic alterations at both the 11p13 and 11p15 regions. To differentiate between the involvement of these two loci, a chromosome 11 was constructed that had one or the other region deleted, and this chromosome was introduced into the tumorigenic Wilms tumor cell line G401. When assayed for tumor-forming activity in nude mice, the 11p13-deleted, but not the 11p15.5-p14.1-deleted chromosome, retained its ability to suppress tumor formation. These results provide in vivo functional evidence for the existence of a second genetic locus (WT2) involved in suppressing the tumorigenic phenotype of Wilms tumor.

Animals↗
PubMed1991

Correlation of the inability to sustain growth in defined serum-free medium with the suppression of tumorigenicity in Wilms' nephroblastoma.

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum-free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175-180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell-mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) chromosome from the nontumorigenic microcell hybrid cells resulted in the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum-free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (EGF) or 5 micrograms/ml of insulin did not alter this effect. However, the addition of 5 micrograms/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum-free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum-free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.

Animals↗
PubMed1991

Suppression of tumorigenicity of a Wilms' tumor cell line is associated with a decrease in synthesis of two proteins.

Wilms' tumor has been associated with deletions in two loci on chromosome 11, and the introduction of a translocated human chromosome [t(X;11)] into a Wilms' tumor cell line (G401.6TG.6) by microcell hybridization suppresses tumor formation in nude mice. The tumorigenic phenotype is restored in segregants of these microcell hybrids, in which the introduced chromosome is lost. We have used ultrahigh-resolution 'giant' two-dimensional gel electrophoresis of metabolically labeled cellular proteins and in vitro translation products of isolated mRNA to identify changes in cellular gene expression that occur in these cell lines. The changes in gene expression associated with these chromosomal manipulations per se are quite minimal. However, we have identified two proteins (p16 and p28) whose synthesis is consistently decreased in three non-tumorigenic (suppressed) microcell hybrid clones relative to parental and segregant tumorigenic lines. They are also decreased at the level of mRNA in at least two of the non-tumorigenic clones. The decrease of these proteins represents markers of the suppressed phenotype, and their down-regulation may conceivably mediate the suppression of tumorigenicity.

Child, Preschool↗
PubMed1990

Irradiation microcell-mediated chromosome transfer (XMMCT): the generation of specific chromosomal arm deletions.

The microcell-mediated chromosome transfer technique has been used to introduce whole chromosomes into malignant cells and revert the tumorigenic phenotype. However, in most instances the limited availability of selectable chromosomes has hindered the ability to reduce the region containing the tumor suppressive information. The work presented here describes a new method to enrich for specific chromosomal arm deletions of selectable chromosomes and thereby more finely focus upon the genetic region of interest. The irradiation-microcell mediated chromosome transfer (XMMCT) technique involves the irradiation of microcells containing single human chromosomes followed by fusion to a nonirradiated host and cytogenetic characterization. The XMMCT procedure was performed on a microcell hybrid containing a der(11) as the only human chromosome. The resultant irradiated microcell hybrids were found to have deletions that ranged from simple interstitial deletions to complex deletions/rearrangements involving only the human der(11) chromosome. The XMMCT procedure has broad applications in generating chromosomal reagents for mapping genetic loci and for use in functional analyses such as tumor suppression studies.

Animals↗
PubMed1989

Serologic properties of the triple antibody-sandwich-lymphocyte-agglutination assay (TASLA).

Detailed studies concerning the serologic properties of the triple antibody-sandwich-lymphocyte-agglutination (TASLA) assay are described herin. The technique is a sensitive one based on sandwiching three layers of antibody onto the target cell. Two different test systems were utilized which included xeno- and allogeneic models. In the xenogeneic test system, rabbit-anti-DA lymphocyte xenosera served as the primary antibody sandwich layer. Goat-anti-rabbit and swine-anti-goat IgG served as the secondary and tertiary antibody sandwich layers, respectively. In the rat allogeneic test system, LEW-anti-BN rat lymphocyte allosera served as the primary antibody layer. Rabbit-anti-rat and goat-anti-rabbit IgG served as the secondary and tertiary antibody sandwich layers, respectively. Several different experiments were run with varying numbers of antibody sandwich layers, and differing concentrations within each layer. The lymphocyte agglutination reaction was then evaluated by regression analysis. Regardless of the number or concentration of antibody sandwich layers, it was found that the reaction could be functionally defined mathematically, by regression analysis. A secondary or tertiary antibody sandwich layer increased assay sensitivity. The level of lymphocyte agglutination was found to be both a linear function of the number of antibody sandwich layers and the concentration of each utilized. In addition, the serological properties of the TASLA assay were extended to the rat allogeneic test system and was again functionally defined mathematically by regression analysis.

Agglutination↗
PubMed1987

Decreased reactivity of allosera against target lymphocytes obtained following thermal injury or long-term cyclosporine treatment.

We speculated that two diverse causes of potent cell-mediated immune suppression, cyclosporine (CsA) and thermal trauma, may demonstrate some similar actions, and thus tested whether either could alter antisera reactivity against allogeneic target lymphocytes. Target splenocytes from 40% body surface area full-thickness burned Brown-Norway (BN) rats demonstrated significant (P = 0.004) decreased reactivity (agglutination) with antisera produced across a full allogeneic barrier (RT1 major histocompatibility complex (MHC) and non-MHC) compared to control splenocytes. Depression of allogeneic splenic target cell reactivity against Lewis (LEW)-anti-BN allosera was similarly observed using lymphocytes from long-term CsA-treated rats (P = 0.004). The decreased reactivity induced by burn trauma was transferable to pooled normal splenocytes or blood lymphocytes by preincubation with burn plasma (P less than 0.001), and was confirmed by a cellular enzyme-linked immunosorbent assay (CELISA) (P = 0.003). In summary, a similarity consisting of decreased antibody reactivity against lymphocytes from either burned or long-term CsA-treated animals was demonstrated. These results suggested that lymphocyte cell surface allogeneic determinants and their expression and/or availability were altered by either regimen.

Animals↗
PubMed1987

Extensive prolongation of rat renal allograft survival following donor or nonspecific transfusions and concomitant immunosuppressant.

We report here a marked beneficial effect upon rat renal allograft survival transplanted across a strong histocompatibility barrier (BN----LEW) by pretransplant concomitant donor-strain blood transfusion (DST) and CsA treatment. Comparisons between recipient groups treated with pretransplant nonspecific blood (NST) and concomitant cyclosporine (CsA) or azathioprine (Aza) administration were also made. LEW recipients receiving only a BN renal allograft survived for a geometric average time of 8.9 days. Recipients receiving 1 ml of donor blood at weekly intervals, each week for three weeks prior to transplantation, demonstrated a geometric mean survival time (GMST) of 40.5 days. Recipients receiving this same regimen and concurrent CsA cover (5 mg/kg/day) starting 7 days prior to the first transfusion with discontinuation 5 days prior to transplantation showed extensive prolongation (greater than 100 days). Recipients treated with only CsA cover survived for a GMST of 34.4 days. LEW recipients receiving 1 ml of nonspecific blood at weekly intervals (DA, BUF, WKY, respectively) each week for 3 weeks prior to transplantation were prolonged to 27.7 days. Recipients treated with this same regimen while under CsA cover also demonstrated extended prolongation (greater than 100 days). Recipients receiving multiple donor blood transfusions under Aza (2 mg/kg/day) cover demonstrated lesser prolongation (22.8 days). Recipients receiving the multiple nonspecific blood protocol under Aza cover showed similar prolongation (38.6 days). Recipients treated only with Aza did not show prolonged survival (9.3 days). These differences in survival were considered significant among the 9 transplant groups as determined by ANOVA (P less than 0.001). The majority of recipient groups showed relatively poor renal function over their life spans, independent of whether prolongation occurred. Yet, renal function in the NST or particularly the DST groups covered by pretransplant CsA, demonstrated the best renal function in our laboratory over many years of investigations using the BN----LEW combination. In conclusion, there was a dramatic synergistic beneficial effect of prior multiple DST or NST specific to CsA, as opposed to another immunopharmacologic agent, Aza.

Animals↗
PubMed1986

Composite tissue (limb) allografts in rats. III. Development of donor-host lymphoid chimeras in long-term survivors.

Eight LEW rat recipients possessing long-term-surviving (206-701 days) LBN vascularized hind limb allografts (CTAs) were tested for donor-host lymphoid chimerism. The recipients received various cyclosporine (CsA) treatment protocols in order to induce indefinite CTA acceptance. Histological examination of long-term-surviving CTAs demonstrated normal-appearing bone marrow in the donor limb. Lymphocytes isolated from host hemopoietic tissues (peripheral blood and/or spleen) by ficoll-hypaque density gradient centrifugation were tested against LEW-anti-BN antisera. Comparisons were made to standard curves employing various known concentrations of LBN and LEW cell combinations. The level of lymphocyte agglutination (dependent variable) showed a significant (P less than 0.025-0.005) linear relationship to the concentration of LBN donor cells (independent variable) present. Lymphocyte suspensions isolated from long-term CTA host peripheral blood and/or spleen showed a mean of 19.7% (+/- 9.7-95% confidence interval) donor LBN mononuclear cells present. Thus, it appeared that lymphoid cells originated from, and/or were released from LBN donor bone marrow into the circulation, resulting in chimeric repopulation of hemopoietic tissues. The presence of donor immunocytes in these limb allograft recipients may have been beneficial, and thus could have helped contribute to the long-term CTA survival observed.

Animals↗