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Generally, stable disease is classified as treatment failure, whereas this may not be the case if tumors of patients with stable disease contain senescent cells, which have lost the capacity to proliferate as a result of treatment.Thus, DNA buy Drotaverine Hydrochloride damageinduced premature cellular senescence may indeed be a relevant factor in determining treatment outcome.Before drug treatment, cells were allowed to attach for h.SN and VP and ethanol, respectively, and stock solutions were diluted in medium just before treatment.For prolonged exposures, medium was replaced every h, and detached cells were harvested and resuspended in the fresh drug medium.Proteins were visualized using the ECL detection system. Cryosections of snapfrozen breast tumor biopsies, from untreated patients or patients who had received neoadjuvant CAF, were fixed for min in formalin in PBS and stained as described above.hydrogen peroxide for min.After being washed in PBS three times, cells were incubated for hwith primary antibody: DO. The sections were washed and exposed to biotinylated goat antimouse antibody for min, followed by streptavidinconjugated horseradish peroxidase for min.After being washed, sections were incubated with diaminobenzidine for min.After a thorough wash with tap water, sections were counterstained with hematoxylin and washed again with tap water.Sections were scored for intensity as follows, no staining, low staining, medium staining, high staining.In these studies we used LST and HCA colon, MCF breast, and A ovarian adenocarcinoma cells, all expressing wildtype p.Growth inhibition after exposure to the drugs was observed in all cell lines investigated.In the LST, MCF, and A cell lines, growth inhibition was characterized by growth arrest, whereas in HCA cells this could be attributed to cell death. The response of LST cells to SN was characterized mainly by prolonged G arrest, although some cells were arrested in the S and GM.A percentage of cells, however, underwent apoptosis, which was maximal after h. In A cells, no apoptosis could be observed by analysis of DNA content and microscopic examination, and cells were predominantly accumulated in G. A similar pattern was observed in MCF cells, although again some cells underwent apoptosis, which peaked at hof treatment, with the cell cycle distribution remaining constant thereafter. In HCA cells, the response to SN treatment was characterized mainly by apoptosis.Contrary to SN exposure, there was little evidence of apoptosis with VP in LST cells either by FACS analysis or by microscopic examination.LST cells were incubated with SN for h, after which the cells were recultured in drugfree medium.There was no growth after drug withdrawal: the cell counts remained constant to weeks, and cells excluded trypan blue at each concentration of the initial SN exposure.Apoptosis could not be observed by flow cytometric analysis of DNA content or cellular morphology, and there was no redistribution of the cell cycle. During treatment, cells exposed to low SN concentrations progressed through G and S phase, followed by accumulation in the GM phase of the cell cycle.Cells exposed to intermediate levels of the drug arrested in late S and GM phase, whereas cells exposed to the highest SN doses were retained in G phase.

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