phosphorus-radioisotopes and Scleroderma--Systemic

Hoechst 33342, but not Hoechst 33258, induces apoptosis and inhibits topoisomerase 1 activity in vivo. Topoisomerase I relaxes superhelical DNA through a single strand breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site, forming a transient intermediate called a cleavable complex. The fate of cellular topoisomerase 1 in Hoechst 33342-induced apoptosis is unknown. We analyzed the binding capacity of topoisomerase 1 to 32P-labeled plasmid pCI DNA, the immunoreactive topoisomerase 1 concentration and topoisomerase 1 activity in BC3H-1 myocytes and HL-60 cells treated with Hoechst 33342 and Hoechst 33258 by using covalent transfer of 32P radioactivity from plasmid DNA to topoisomerase 1, Western blotting and topoisomerase 1-mediated plasmid relaxation assay, respectively. Hoechst 33342, but not Hoechst 33258, induced topoisomerase 1 dysfunction in both BC3H-1 myocytes and HL-60 cells measured by (1) a decrease in the topoisomerase 1 to DNA binding capacity or cleavable complex formation; (2) a decrease in intracellular concentration of immunoreactive topoisomerase 1; and (3) an inhibition of nuclear endogenous topoisomerase 1 activity. These results suggest that destruction of immunoreactive topoisomerase 1 and topoisomerase 1-DNA complexes or cleavable complexes results in inhibition of topoisomerase 1 activity, a key step in the Hoechst 33342-induced apoptotic process.

Topics: Animals; Apoptosis; Autoantibodies; Benzimidazoles; Bisbenzimidazole; Blotting, Western; Cell Line; Cell Nucleus; DNA; DNA Topoisomerases, Type I; DNA, Superhelical; HL-60 Cells; Humans; Mice; Muscles; Phosphorus Radioisotopes; Plasmids; Scleroderma, Systemic

We studied sera of 371 consecutive new patients with systemic sclerosis (SSc; scleroderma) who were first evaluated during 1984-1988. All sera were tested for antinuclear antibodies by immunofluorescence staining using HEp-2 cells as substrate. We excluded 219 sera showing dark nucleoli and screened for antibodies to Th in the remaining 152 sera by immunoprecipitation of a 32P-labeled HeLa cell extract. Fifteen (4.0%) of 371 sera were anti-Th+. Anti-Th antibodies were present in 14 (8.4%) of 167 SSc patients with limited cutaneous involvement, in 1 of 167 with diffuse cutaneous involvement, and in 0 of 37 with SSc overlap syndrome. Among 244 controls with other connective tissue diseases, anti-Th was detected in only 3 patients, all having primary Raynaud's phenomenon of less than 2 years duration. In the subgroup with SSc with limited cutaneous involvement, the 14 anti-Th+ patients had a significantly greater frequency of puffy fingers, small bowel involvement, and hypothyroidism, and a significantly lower frequency of arthralgia and/or arthritis. Their cumulative survival rate from the time of onset of symptoms was lower than that for anti-Th- patients (78% versus 91% at 10 years), primarily due to 3 deaths from pulmonary arterial hypertension (2 from primary pulmonary hypertension and 1 from pulmonary hypertension secondary to pulmonary interstitial fibrosis). Serum anti-Th antibodies are present almost exclusively in patients with SSc with limited cutaneous involvement or in those with primary Raynaud's phenomenon whose disease may evolve to SSc with limited cutaneous involvement, and these antibodies may identify those patients who are at greater risk for reduced survival.

Topics: Autoantibodies; Fluorescent Antibody Technique; Humans; Phosphorus Radioisotopes; Precipitin Tests; Ribonucleoproteins; Ribonucleoproteins, Small Nuclear; RNA; Scleroderma, Systemic