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C A Reznikoff

Publications and source records attributed to C A Reznikoff.

66 records · Page 4Linked to original sources
PubMed1985

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells.

As a prelude to study the promotion with TPA of in vitro transformation of human urothelial cells (HUC) in culture, we characterized tumor promoter TPA receptors in primary cultures of HUC. [3H]TPA bound specifically to intact living HUC; maximum specific binding was attained in approximately 30 min at 37 degrees C. [3H]TPA bound to HUC in a saturable and competitive manner. Scatchard analysis of specific binding to intact cells displayed a single slope corresponding to an equilibrium dissociation constant (Kd) of 0.56 nM; at saturation TPA-binding capacity was 2.37 pmol/10(6) HUC (1.43 X 10(6) sites per cell). [3H]TPA bound specifically and with high affinity to the particulate fractions of HUC; binding was both saturable and reversible. Saturation of the specific binding of [3H]TPA occurred at approximately 1 nM at 4 degrees C. Scatchard analysis of specific binding to the particulate fraction displayed a single slope corresponding to a Kd of 1.08 nM; at saturation TPA-binding capacity was 2.05 pmol/mg protein (750 000 molecules per HUC). [3H]TPA binding was inhibited by the biologically active phorbol ester, phorbol didecanoate, whereas inactive phorbol did not compete for TPA binding. Binding was not affected by sodium saccharin, epidermal growth factor, retinoic acid or dexamethasone. [3H]TPA bound specifically to the HUC cytosolic fraction but only in the presence of calcium and phosphatidylserine. Calcium-activated and phospholipid-sensitive protein kinase activity was detected in HUC fractions. These results indicate the presence of high-affinity specific receptors for TPA in HUC.

Caenorhabditis elegans Proteins↗
PubMed1984

Cultures of normal human urothelial cells from ureters of perfused cadaver transplant kidneys.

In this comparative study, we report that urothelial cultures initiated from ureters of mechanically perfused cadaver-obtained transplant kidneys display characteristics of explant attachment, explant viability, cellular morphology and area of outgrowth essentially indistinguishable from cultures established from ureters of living donor kidneys. We conclude that ureter specimens from cadaver kidneys are a suitable, routinely available source of tissue from which cultures of normal human urothelial cells can be propagated.

Cadaver↗
PubMed1984

Bioconversion and macromolecular binding of 2-amino-4-(5-nitro-2-furyl)thiazole by cultured rat urothelial cells.

Bioconversion and binding of 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT) were examined using cultured rat bladder epithelial cells from weanling male F344 rats. Bladder cells were obtained in large quantities from outgrowths of dissected explants which were grown on collagen gels. Metabolic potential of rat urothelial cells to activate ANFT was evaluated by incubating primary culture cells with [2-14C]ANFT for 48 hr. Metabolites were subsequently analyzed by chromatographic and spectroscopic methods. Thin-layer chromatography of the ethyl acetate:diethyl ether (1:1, v/v) extract of the culture medium revealed two regions of radioactivity with Rf values of 0.12 and 0.60, the former corresponding to ANFT and the latter to one of its metabolites. High-pressure liquid chromatography of the solvent extract revealed two major peaks, with retention times of about 4 and 9 min, corresponding with the metabolite and ANFT, respectively. Low-resolution mass spectrum of the isolated metabolite showed a molecular ion at m/e 181. The metabolite was identified as 1-[4-(2-aminothiazolyl)]-3-cyano-1-propanone based on its chromatographic and spectral characteristics in comparison with the synthetic compound. About 24% of the recovered radioactivity from the culture medium was extractable into the organic phase, a majority of which was identified as 1-[4-(2-aminothiazolyl)]-3-cyano-1-propanone. Analysis of binding to proteins and nucleic acids prepared following exposure of [2-14C]ANFT revealed a 15- and 9-fold greater amount of binding, respectively, in cultures incubated with bladder cells than their corresponding heat-inactivated controls. Furthermore, homogenates of cultured bladder cells reduced ANFT on anaerobic incubation with reduced nicotinamide adenine dinucleotide phosphate to generate 1-[4-(2-aminothiazolyl)]-3-cyano-1-propanone. On reduction of [2-14C]ANFT with rat bladder or liver homogenates, about 23 and 11%, respectively, of the initial amounts of radioactivity were bound to the trichloracetic acid-insoluble fraction. These data demonstrate that rat bladder cells possess the metabolic capability to reduce ANFT and to generate reactive intermediate(s) that bind to cellular macromolecules.

Animals↗
PubMed1983

Growth and characterization of normal human urothelium in vitro.

A method for initiating rapidly growing cultures of normal human transitional cells from ureter and embryonic bladder specimens has been developed and quantified. A new microdissection technique was used to nonenzymatically separate the urothelium. The use of enriched medium containing 10 micrograms/ml insulin, 5 micrograms/ml transferrin, and 1 microgram/ml hydrocortisone resulted in improved growth. The use of thin collagen gel substrates (0.6 ml/60 mm petri dish) resulted in 97% attachment of explants compared to 77% attachment on plastic. Explants grown on thicker collagen (2 ml/60 mm petri dish) showed, in addition to better attachment, enhanced growth of cells as determined both by measurements of colony size and cell density. Cultures of transitional cells that were initiated using explants could be passed three to five times using 0.1% EDTA for dispersion. Autoradiography of [3H]thymidine-labeled cells showed an initial phase of rapid cell division in primary explant cultures and restimulation of cell division in passaged cultures. Transmission electron microscopy showed that the cells growing out from the explants were continuous with the stratified urothelium maintained in the original explant. Stratification of transitional cells occurred in cultures of both ureter and embryonic bladder cells. Surface cells were joined near their apices by junctional complexes. Desmosomes and Golgi vesicles were present in all cells. Passage in culture did not alter the morphological characteristics of cells.

Cell Division↗
PubMed1983

Altered growth patterns in vitro of human papillary transitional carcinoma cells.

In vitro growth patterns and morphologic characteristics of five low-grade human papillary transitional cell carcinomas (TCCs) were compared and contrasted with those of normal human urothelial cells in culture. Biopsies of TCC were performed by transurethral resection. Specimens of normal human ureters were obtained surgically. Singly dispersed TCC cells grew in 0.3% agarose semisolid medium with a cloning efficiency ranging from 0.02% to 0.71%. Singly dispersed normal ureteral urothelial cells under the same conditions did not form colonies in 0.3% agarose. Neither singly dispersed TCC nor normal urothelial cells formed colonies when plated on collagen-gel substrates. In primary explant culture, normal human urothelial cells grew rapidly, to form tightly adherent flat sheets of apparently nonmotile cells. Autoradiographic labeling with 3H-thymidine of growing cultures of normal urothelial cells showed cell division primarily in the zones of growth near the explant. Outgrowth of TCC from primary explants was loosely adherent. One TCC explant culture gave rise to a continuous suspension culture. Numerous multilayered cellular formations of fronds, nodules, and "walls" were seen around the periphery of TCC explant colonies. Autoradiography showed that these multilayered areas of TCC growth contained actively dividing cells. The altered ability of papillary TCC to form superficial multilayered formations in vitro distinguishes them from normal human urothelium and reflects the morphologic characteristic of this tumor type in vivo.

Adolescent↗
PubMed1981

In vitro chemical mutagenesis and viral transformation of a human endothelial cell strain.

We have established and characterized a diploid cell strain of normal human endothelial cells, RuBa 7E. RuBa 7E cells have an average cloning efficiency of 20% during early passages and undergo approximately 50 doublings in vitro before senescing spontaneously. At confluence, RuBa 7E cells form a homogeneous monolayer of flat polygonal cells. RuBa 7E cells react positively with antibody to human endothelial-specific Factor VIII. The toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine on RuBa 7E cells were studied and are similar to those reported for diploid human fibroblasts. Mutant cells lacking hypoxanthine-guanine phosphoribosyltransferase were selected by their resistance to 6-thioguanine. The spontaneous incidence of mutants was less than or equal to 6 X 10(-6), and the induced incidence was 4.4 X 10(-4) at a survival frequency of 0.05. All 17 mutants that were tested lacked detectable hypoxanthine-guanine phosphoribosyltransferase activity, and none grew in medium containing azaserine and hypoxanthine. Autoradiography showed that mutant cells incorporated radioactive adenine but did not incorporate radioactive hypoxanthine. Unlike human fibroblasts, in which the recovery of 6-thioguanine-resistant mutants is reduced by contact feeding when the inoculum size during selection is increased above 10(4) cells per P60 dish, 5 to 10 X 10(4) RuBa 7E cells can be plated per P60 dish without reducing mutant recovery. This apparent lack of plating density suppression of mutant recovery makes RuBa 7E cells a comparatively compact and economical system for quantifying mutagenesis in diploid human cells. In order to determine whether RuBa 7E cells would undergo a distinct morphological transformation toward cancer in vitro, we infected them with SV40. As early as 14 days postinfection, discrete foci of morphologically transformed, mitotically active cells were seen against a monolayer background of normal cells when cultures were maintained in medium with low serum. Seven of the 33 foci which were obtained were studied for SV40-specific viral T-antigen, and all were positive. The facility with which RuBa 7E cells can be mutagenized and the ease with which morphological transformants can be identified make these cells potentially useful for studies comparing the mutagenic and transforming effects of chemicals and other agents on diploid human cells.

Cell Division↗
PubMedDate not supplied

Correlation between N-acetyltransferase activities in uroepithelia and in vivo acetylator phenotype.

The relationship between in vivo acetylator phenotype of individuals and N-acetyltransferase (NAT) activity in the cytosol of their cultured uroepithelia was examined in four urology patients. In vivo acetylator phenotypes were assigned by determining the ratio of N-acetyl vs. total [N-acetyl+free] sulfamethazine in urine and blood following a single oral dose (1 gm) of sulfamethazine. From the same patients, a surgical specimen of the ureter was obtained, uroepithelial cells were cultured in vitro, and the cytosols prepared. NAT activities were determined by measuring the amount of 4-acetylaminobiphenyl formed from incubation of uroepithelial cytosol with the substrate, 4-aminobiphenyl, and the cofactor [14C]acetyl coenzyme A. The two individuals phenotyped as "slow acetylators" by the in vivo method had NAT activities of 8.3 and 16.2 pmol 4-acetylaminobiphenyl/mg protein/min. In contrast, the two individuals phenotyped as "rapid acetylators" showed activities of 50.9 and 109.5 pmol 4-acetylaminobiphenyl/mg protein/min. The rapid acetylators exhibit about 6-fold greater uroepithelial NAT activities than slow acetylators, thus showing a direct correlation between the NAT activity in the uroepithelium, the target tissue of the human bladder carcinogen 4-aminobiphenyl, and the in vivo acetylator phenotype. These results imply that susceptibility of individuals to arylamine-induced bladder cancer might be associated with NAT activities in their target cells and that in vivo acetylator phenotyping could serve as a useful and relevant biochemical screening marker to assess the risk of developing bladder cancer.

Acetylation↗
PubMedDate not supplied

Relationship between in vivo acetylator phenotypes and cytosolic N-acetyltransferase and O-acetyltransferase activities in human uroepithelial cells.

The in vivo acetylator phenotype as well as N-acetyltransferase (NAT) and O-acetyltransferase (OAT) activities in cytosols from cultured uroepithelia were determined in 25 urological patients. In vivo acetylator phenotypes were categorized by determining the amounts of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine in urine after the administration of a 200 mg dose of caffeine. Subjects were grouped according to the AFMU/AFMU + 1-methylxanthine ratio as "slow" (< 0.41) or "rapid" (> or = 0.41) acetylators. The uroepithelia were obtained from these subjects, cultured in vitro, and the cytosols were prepared. NAT and OAT activities were determined using 4-aminobiphenyl (ABP) and [3H]N-hydroxy-4-aminobiphenyl (N-OH-ABP) as substrates, respectively. In vivo phenotyping resulted in 18 patients being slow acetylators and seven rapid. The mean NAT and OAT activities for these different subsets were: 3.58 nmol/mg protein/min and 409 pmol/mg tRNA/mg protein for the slow; and 3.38 nmol/mg protein/min and 428 pmol/mg tRNA/mg protein for the rapid, respectively. Furthermore, in individual samples, NAT and OAT activities tended to parallel each other, implying that the same enzyme might catalyze both NAT and OAT activities in uroepithelia. The N-acetylation of ABP was inhibited by N-OH-ABP and also by p-aminobenzoic acid, a substrate which is preferred by the monomorphic NAT enzyme. Similarly, OAT-mediated binding of [3H]N-OH-ABP to tRNA was inhibited in a dose-dependent manner by ABP and p-aminobenzoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗