RESUMEN
Wolbachia influence the fitness of their invertebrate hosts. They have effects on reproductive incompatibility and egg production. Although the former are well characterized, the mechanistic basis of the latter is unclear. Here, we investigate whether apoptosis, which has been implicated in fecundity in model insects, influences the interaction between fecundity and Wolbachia in the planthopper Laodelphax striatellus. Wolbachia-infected females produced about 30% more eggs than uninfected females. We used the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling staining to visualize apoptosis. Microscopic observations indicated that the Wolbachia strain wStri increased the number of ovarioles that contained apoptotic nurse cells in both young and aged adult females. The frequency of apoptosis was much higher in the infected females. The increased fecundity appeared to be a result of apoptosis of nurse cells, which provide nutrients to the growing oocytes. In addition, cell apoptosis inhibition by caspase messenger RNA interference in Wolbachia-infected L. striatellus markedly decreased egg numbers. Together, these data suggest that wStri might enhance fecundity by increasing the number of apoptotic cells in the ovaries in a caspase-dependent manner. Our findings establish a link between Wolbachia-induced apoptosis and egg production effects mediated by Wolbachia, although the way in which the endosymbiont influences caspase levels remains to be determined.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Hemípteros/fisiología , Wolbachia/fisiología , Secuencia de Aminoácidos , Animales , Caspasas/genética , Femenino , Fertilidad , Aptitud Genética , Hemípteros/microbiología , Ovario/microbiología , Ovario/fisiologíaRESUMEN
HL-60 cells that stably express transfected wild-type (wt) p53 were used to determine whether restoration of wt p53 increased the chemosensitivity of cells that normally lack p53 activity. The wt p53 HL-60 transfectants (SN3 cells) were more sensitive than the parental (S) cells to a number of common anticancer drugs representing various mechanisms of action, whereas HL-60 cells transfected with p53 genes mutated at codons 248 and 143 were not sensitized. The sensitization ratio due to the transfected wt p53 varied from about 2-fold for cisplatin to over 50-fold for thymidine. Cells treated with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) were used to study changes in various p53-associated gene expressions. A higher percentage of apoptotic cells among the SN3 cells was observed than among the S cells at each concentration of FdUrd. The S cells had undetectable levels of bax and high levels of bcl-2, whereas the SN3 cells had undetectable levels of bcl-2 levels and appreciable basal levels of bax. After FdUrd treatment of SN3 cells, both p53 and bax levels increased, but the induction of bax was faster than that of p53 and paralleled the appearance of apoptotic DNA laddering. FdUrd treatment induced p21 expression and increased the G1 fraction of the SN3 cells but did not induce p21 or change the phase distribution in the S cells. FdUrd treatment also induced the expression and phosphorylation of cyclin D1 in the SN3 cells but not in the S cells. These results show that transfected wt p53 confers multidrug sensitivity to HL-60 cells by re-adjustment of the expressions of apoptosis genes and displays other properties characteristic of endogenously originated wt p53.
Asunto(s)
Resistencia a Múltiples Medicamentos/genética , Fluorodesoxiuridilato/farmacología , Genes p53/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Resistencia a Antineoplásicos/genética , Genes bcl-2/fisiología , Células HL-60/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Transfección , Proteína X Asociada a bcl-2RESUMEN
HL60 cells, which lack the p53 gene due to a deletion, were used as an in vitro model system to study the effect of wild-type p53 gene expression on hematopoietic differentiation. We transfected HL60 cells with wild-type p53 and two mutant p53 cDNAs encoding the Val to Ala mutation at codon 143 and the Arg to Trp mutation at codon 248. Flow cytometry, growth, and cytochemical analysis for alpha-napthyl butyrate esterase activity and nitroblue tetrazolium reduction indicated that wild-type p53 but not mutant p53 induced early monocytic differentiation in the transfected HL60 cells without terminal growth arrest. The wild-type p53 transfectants did not differentiate along the granulocytic pathway, even when induced with 1.25% DMSO for 6 days; rather, these cells resembled monocytic cells, confirming that wild-type p53 transfection caused these cells to become committed to differentiate along the monocytic pathway. HL60 cells transfected with wild-type p53 were more sensitive to stress, such as growth in serum-depleted medium and exposure to a chemotherapeutic agent, etoposide.