STUDIES OF THE MOLECULAR MECHANISMS OF CHROMOSOME ABERRATION FORMATION (DNA REPAIR, CROSSLINKS, MITOMYCIN C)

The objective of this research has been to study the molecular basis for chromosome aberration formation. Predicated on a variety of data, Mitomycin C (MMC)-induced DNA damage has been postulated to cause the formation of chromatid breaks (and gaps) by preventing the replication of regions of the genome prior to mitosis. The basic protocol for these experiments involved treating synchronized Hela cells in G(,1)-phase with a 1 (mu)g/ml dose of MMC for one hour. After removing the drug, cells were then allowed to progress to mitosis and were harvested for analysis by selective detachment. Utilizing the alkaline elution assay for DNA damage, evidence was obtained to support the conclusion that Hela cells can progress through S-phase into mitosis with intact DNA-DNA interstrand crosslinks. A higher level of crosslinking was observed in those cells remaining in interphase compared to those able to reach mitosis at the time of analysis. Dual radioisotope labeling experiments revealed that, at this dose, these crosslinks were associated to the same extent with both parental and newly replicated DNA. This finding was shown not to be the result of a two-step crosslink formation mechanism in which crosslink levels increase with time after drug treatment. It was also shown not to be an artefact of the double-labeling protocol. Using neutral CsCl density gradient ultracentrifugation of mitotic cells containing BrdU-labeled newly replicated DNA, control cells exhibited one major peak at a heavy/light density. However, MMC-treated cells had this same major peak at the heavy/light density, in addition to another minor peak at a density characteristic for light/light DNA. This was interpreted as indicating either: (1) that some parental DNA had not been replicated in the MMC treated sample or; (2) that a recombination repair mechanism was operational. To distinguish between these two possibilities, flow cytometric DNA fluorescence (i.e., DNA content) measurements of MMC-treated and control cells were made. These studies revealed that the mitotic cells that had been treated with MMC while in G(,1)-phase displayed a 10-20% lower DNA content than untreated control cells when measured under conditions that neutralize chromosome condensation effects (i.e., hypotonic treatment). These measurements were made under conditions in which the binding of the drug, MMC, was shown not to interfere with the stoichiometry of the ethidium bromide-mithramycin stain. At the chromosome level, differential staining techniques were used in an attempt to visualize unreplicated regions of the genome, but staining indicative of large unreplicated regions was not observed. These results are best explained by a recombinogenic mechanism. A model consistent with these results has been proposed.^

ATP- and carbachol -stimulated 1,2-diacylglycerol production during sensitization of adenylyl cyclase

Stimulation of LM5 cells with the phorbol ester 4$\beta$-phorbol 12-myristate 13-acetate (PMA), causes a 2-4 fold sensitization of hormonally-stimulated adenylyl cyclase (AC) activity. This effect is thought to be due to protein kinase C (PKC)-mediated phosphorylation of either G$\sb{\rm i}$ or the catalytic subunit of AC. PKC are components of the phosphatidylinositol-4,5-bisphosphate phospholipase C (PIP$\sb2$-PLC) pathway. The currently accepted model of this pathway is that its activation by an agonist results in the production of inositol 1,4,5-triphosphate (IP$\sb3$) which causes Ca$\sp{++}$ mobilization, and 1,2-diacylglycerols (DAG) which activate PKC. Based on this model, we predicted that stimulation of purinergic and muscarinic receptors with the agonists ATP and carbachol (CCh), respectively in the LM5 cells, should sensitize AC. Surprisingly we found that only stimulation of the purinergic receptors in these cells caused a sensitization of PGE$\sb1$-stimulated AC measured in cell-free assays.^ We hypothesized that ATP-and CCh-stimulated differential DAG production contributes to the effectiveness of these two agonists to sensitize PGE$\sb1$-stimulated AC activity. To test this hypothesis directly, we performed a combined high-performance liquid chromatography and gas-liquid chromatography analysis of the DAG produced in the LM5 cells in response to stimulation with ATP and CCh.^ We found that both ATP and CCh increased levels of 23 species of DAG. Relative to the control levels (0.261 nmol DAG/100 nmol phospholipid) the CCh-induced increase in DAG levels was 280% (0.738 $\pm$ 0.051 nmol DAG/100 nmol phospholipid) whereas the ATP-induced levels increased 180% (0.441 t 0.006 nmol DAG/100 nmol phospholipid). Neither agonist created new species or eliminated the existing ones. The major species which comprised $\approx$50% of the total cellular DAG in all of the groups were 16:0-18:1, 18:0-18:1, 18:1-18:1, and 18:0-20:4. CCh was more effective than ATP at stimulating these major DAG species.^ It is concluded that factor(s) other than DAG contribute(s) to the differences between ATP-and CCh-sensitization of PGE$\sb1$-stimulated AC activity in the LM5 cells. ^