Apoptosis of vascular smooth muscle cells. Protein kinase C and oncoprotein Bcl-2 are involved in regulation of apoptosis in non-transformed rat vascular smooth muscle cells.

We examined the effect of several inhibitors/activators of various protein kinases on the proliferation and apoptosis of nontransformed rat coronary vascular smooth muscle cells (SMC). As expected, all the compounds (calphostin C, KT5720, KT5823, verapamil, W7, and dibutyryl-cAMP) inhibited SMC proliferation, as judged by [3H]thymidine incorporation. Three (calphostin C, verapamil and dibutyryl-cAMP) of the six compounds caused occurrence of the classical apoptotic morphology in SMC. The effect of calphostin C, an inhibitor of protein kinase C, was examined in more detail due to the known involvement of this kinase in regulation of apoptosis in a variety of cell types. In SMC cultures exposed for 1, 2, and 3 days to 0.1 mumol/L calphostin C, 7 +/- 1%, 32 +/- 3%, and 29 +/- 3% of cells underwent apoptosis, respectively, as assessed by cell morphology (control cultures had 1 to 3% of apoptotic cells). The effect of calphostin C was transient in that on day 6 following exposure to this compound the number of apoptotic cells declined to control values. Simultaneous with the induction of apoptotic morphology in SMC, a decline was seen (within 24 hours) in expression of the oncoprotein Bcl-2 in morphologically nonapoptotic SMC. An altered distribution of Bcl-2 was seen in the apoptotic cells. The calphostin C-induced generation of apoptotic cells in SMC cultures and the decline/alteration of Bcl-2 expression were not accompanied by degradation of DNA into nucleosomal fragments. In conclusion, normal, nontransformed rat coronary artery vascular SMC undergo apoptosis when exposed to an inhibitor of protein kinase C (calphostin C), to a calcium channel blocker (verapamil), and to a stimulator of cAMP-dependent protein kinase (dibutyryl-cAMP). The induction of apoptosis by the inhibitor of protein kinase C is accompanied by alterations in the Bcl-2 expression but not by DNA fragmentation.

Onset of chromatin fragmentation in chloroma cell apoptosis is highly sensitive to UV and begins at non-B DNA conformation.

UV irradiation induces programmed death, or apoptosis, in rat chloroleukaemia cells, a process characterized by typical cell morphology, fragmentation of chromatin into units of single and multiple nucleosomes, and transcriptional activation of LINE. Our study shows that this process is initiated by very low UV doses: exposure to one minimal erythema dose (MED, about 200 J/m2), at defined wavelengths ranging from 270 to 330 nm, is sufficient. By sequencing 100 randomly selected blunt-end chromatin fragments (single and multiple nucleosomes) we observed that at an early stage of apoptosis the fragmented DNA contains a four-fold excess of both long and short interspersed nuclear retroelements (LINEs and SINEs). A distinct sequence finding was also the observation that fragmented DNA is very rich in microsatellites, (CA)n dinucleotides in particular, and in long stretches of homopurine/homopyrimidine domains. The present results thus indicate that chromatin fragmentation in UV-induced apoptosis begins at non-random locations involving non-B DNA conformation, and that the onset of the suicide process in chloroleukaemia cells is surprisingly sensitive to UV exposure.

Isolation and characterization of a substitution mutation in the ompR gene of Salmonella typhimurium LT2.

The expression of the genes ompC and ompF encoding major outer membrane proteins is dependent on the ompR-envZ operon. Here we describe the isolation and characterization of an ompR mutation, a single-base-pair change, that results in an Arg-to-Cys substitution. When present in multiple copies, the mutant allele conferred a dominant OmpC- OmpF+ phenotype. Furthermore, the mutant allele exhibited allele-specific negative complementation with other ompR mutations. This ability, together with its dominant character, suggested that the OmpR protein is capable of multimerization.