Colocalization of laminin and fibronectin in bovine lens epithelial cells in vitro.

The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse intracellular patterns for type IV collagen and entactin, observed to a similar degree throughout the colonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cell surfaces and in between cells in the densely packed center of the colonies, and as "adhesive plaques" and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin.

Cellular recovery of dividing and confluent C3H10T1/2 cells from N-methyl-N'-nitro-N-nitrosoguanidine in the presence of ADP-ribosylation inhibitors.

The relationship between treatment with 3-methoxy-benzamide (MBA), a potent inhibitor of ADP-ribosylation reactions, and the response of C3H10T1/2 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been examined. Quiescent cells effected potentially lethal damage repair (PLDR) over a 48-h period following MNNG and the repair was coincident with the removal of DNA strand breaks. MBA had no effect on PLDR but was very co-cytotoxic with MNNG in dividing cells. The presence of MBA caused the appearance of an additional number of DNA strand breaks following MNNG in both quiescent and dividing cells. These results suggest that ADP-ribosylation is required for normal cell cycle progression following DNA damage in dividing cells.

Effect of nicotinamide analogues on recovery from DNA damage in C3H10T1/2 cells.

The effects of nicotinamide analogues on cellular recovery following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment have been characterized in the transformable cell line, C3H10T1/2. The recovery of cell division potential was measured under conditions which allow simultaneous quantification of intracellular levels of poly(adenosine diphosphate ribose), nicotinamide adenine dinucleotide, and rates of RNA, DNA, and protein synthesis. 3- Methoxybenzamide (MBA), 3-aminobenzamide, and benzamide, which are effective inhibitors of adenosine diphosphate ribosyltransferases , blocked recovery of cell division following treatment with 34 microM MNNG, while the noninhibitors , 3- methoxybenzoate and benzoate, had no effect. In the presence of MBA, cells progressively lost the ability to resume cell division during the first 24 to 36 hr following DNA damage. The intracellular levels of poly(adenosine diphosphate ribose) increased approximately 7-fold within 20 min following MNNG treatment, and 1 mM MBA inhibited this increase by approximately 82%. In the presence of MBA, a dramatic decrease in the rate of DNA synthesis occurred approximately 16 hr after MNNG treatment, while RNA and protein synthesis continued at rates similar to those in cells treated with MNNG alone.

Mono- and poly(ADP-ribose) metabolism following DNA damage.

Newly developed chemical methods have been applied to study ADP-ribosyl transferase reactions in intact cells following DNA damage. The intracellular levels of NAD and protein-bound monomeric and polymeric ADP-ribose residues were measured in cultured human cells following UV irradiation and in cultured mouse cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). UV irradiation of cells caused a rapid increase in the levels of poly(ADP-ribose). Individual molecules of the polymer are present only transiently and the overall rate of conversion of NAD to poly(ADP-ribose) is proportional to the cellular content of DNA strand breaks. Treatment of cells with MNNG also causes a rapid increase in the levels of both monomers and polymers of ADP-ribose. Non-toxic levels of members of two different classes of ADP-ribosyl transferase inhibitors prevent recovery of cell division following treatment of C3H10T1/2 cells with MNNG, while closely related compounds that are not inhibitory have no effect. These studies demonstrate that DNA damage results in a rapid perturbation of ADP-ribose metabolism and suggest that ADP-ribosyl transferase activity is necessary for cellular recovery from DNA damage.

Reactivity in vivo of the carcinogen N-hydroxy-2-acetylaminofluorene: increase by sulfate ion.

Injections of sulfate ion in rats given the carcinogen N-hydroxy-2-acetylaminofluorene increased (i) the formation of 1-and 3-(methion-S-yl)-2-acetylaminofluorene bound to protein in the liver, (ii) the formation of fluorenyl derivatives bound to total protein, ribosomal RNA, and DNA in the liver, and (iii) the toxicity of the carcinogen. These data provide evidence that the highly reactive ester 2-acetylaminofluorene-N-sulfate, previously suggested as an ultimate reactive and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene, is formed in the rat liver in vivo.