An Immunocytochemical Analysis Of Proliferation, Apoptosis And Expression Of P53 Protein In Leukemia Cells Of Adult T-cell Leukemia

IntroductionIn order to see the relationship of proliferation and apoptosis in leukemia cells and effects of p53 protein expression on them, an immunohistochemical and morphometric analysis of leukemia cells was made on the formalin - fixed paraffin - embedded peripheral blood tissue specimen ( PBTS) of 20 leukemia cases (7 cases of chronic type adult T -cell leukemia (ATL) , 7 cases of acute type, 4 cases of B - cell chronic lymphocytic leukemia and 2 cases of acute my-elogenous leukemia). There were many nuclei labeled by the anti - Ki67 antibody , suggesting an entering proliferation cycle of leukemia cells out of the bloodstream. The number of cases presenting p53 protein labeled by anti - p53 - DO7 antibody was significantly higher in acute type ATL than in the others. The leukemia cells expressing p53 protein labeled by anti - p53 - DO7 antibody were significantly smaller than those without expression of p53 protein, suggesting a helerogenous feature of leukemia cells in acute type ATL. Most leukemia cells were medium - sized. There were 5 cases of ATL revealing a few leukemia cells expressing p53 protein phosphorylated at serine 392 that were labeled by the anti - p53 - PHOS antibody. There was a co - relation between the labeling of Ki67 antigen and the appearance of the cleave caspase -3 (p=0.0175) in spite of a lack of relation between the expression of p53 protein and the appearance of cleaved caspase - 3, suggesting a molecular mechanism bridging proliferation and apoptosis in ATL cells.Materials and methodsFormalin - fixed and paraffin - embedded peripheral blood tissue specimens ( PBTSs) of 7 patients with acute type adult T - cell leukemia (ATL) , 7 patients with chronic type ATL, 4 patients with chronic B - cell leukemia, and 2 patients with myelogenous leukemia were incorporated. The immunohistochemi-cal method and morphometry method were used to analyze the data.ResultsMany or most nuclei were labeled by means of the polymer method of the anti - Ki67 antibody. In most cases, p53 - DO7 labeled many cells in the three antibodies. However, the three antibodies consistently showed that p53 protein was expressed in neither more nor less leukemia cells in acute type ATL. There were significant differences in the number of cases with leukemia cells labeled by p53 - DO7 between acute type and chronic type ATL (p =0.01456) and acute type ATL and cases with B - CLL and AML (p =0. 0250). The immunostain of the anti - cleaved caspase - 3 antibody was noted in the cytoplasm of leukemic cells. Evaluation of the immunostain of the anti - cleaved caspase - 3 over the grade 2 was seen especially in acute type ATL cases. There were co - relations between the labeling of Ki67 antigen and the appearance of cleave caspase - 3 ( p =0. 00877) in spite of no relation between the expression of p53 protein and the appearance of the cleaved caspase - 3. The immunostain of the anti - ssDNA antibody was dense and was seen on many nuclei. In the examined cases except one case of AML, many or most nuclei of the leikemia cells were labeled. There was no corelation between the appearance of cells expressing cleaved caspase -3 and the labeling of the anti - ssDNA antibody, indicating that the appearance of a large amount of single - stranded DNA in the nuclei of the leukemia cells was the result of DNA degeneration in preparing or storing PBTS. Morphometric analysis of nuclei indicated a significantly smaller area factor and form factor in the leukemia cells expressing p53 protein labeled by p53 - DO7 than those with-out p53 expression. There were no significant differences in nuclear length, width, area factor and form factor among chronic type ATL. acute type ATL, B - CLL and AML.DiscussionIt is widely known that the Ki67 antigen is expressed in proliferating cells in the cell cycle phases from late Gl to M . The presented study reported that most leukemia cells labeled by the anti - Ki67 antigen antibody have a potential to re - enter the proliferation cycle when going out of the peripheral bloodstream.This study suggests that p53 protein expressed in leukemia cells was mutant because the immunostain of p53 - DO7 and p53 - 1801 was seen on nuclei of leukemia cells. It is well accepted that p53 protein over - expressed on nuclei is a result of the mutant p53 protein. A significantly high number of acute type ATL with expression of p53 protein labeled by DO7 was reported, suggesting a genetic alteration of p53 in the transition from chronic type to acute type ATL. While phosphorylation of p53 at serines 15 and 392 found in ATL is critical for complex formation, p53 - Phos that labels phosphorylation of p53 at serine 392 labeled only a few cells in a mere 5 ATL cases, suggesting that physiological expression of the p53 protein with phosphorylation at serine 392 could not detected by means of the polymer method. This study reported that leukemia cells expressing p53 protein labeled by p53 - DO7 had significantly smaller nucleus area factor than those without expressions of the p53 protein, and therefore that the leukemia cells in acute type ATL were heterogenous.Cleaved caspase - 3 is an effector caspase and the trigger for the cascade to apoptosis, which activates DNA fragment factors (DFF) that degenerate chro-matin. The appearance of the cleaved caspase - 3 is then able to predict apoptosis. This study indicated that there was a co - relation between the expression of Ki67 antigen and the expression of cleaved caspase -3. ,Single - stranded DNA appears in apoptosis, DNA folks of DNA synthesis and DNA - dependent RNA synthesis. Detection of ssDNA by a low sensitivity...