Differentiated Effect Of Mda-7/IL-24 And Alternative Splice Variant Of Mda-7/IL-24 (mda-7/IL-24as1) On Hematopoietic Malignances

Melanoma differentiation associated gene-7(mda-7) was isolated by Jiang et al from a melanoma cell line induced to differentiate by interferon -βand mezerein.On the basis of its structure,cytokine-like properties,and proposed mode of action, mda-7 has now been classified as IL-24.Since its initial isolation from melanoma ceils,the mda-7/IL-24 gene has been studied in numerous other solid tumor types, including carcinoma of the lung,breast,prostate and cervix,consistently demonstrating growth-inhibitory effects on various tumor types.Although mda-7/IL-24 has been a known candidate cancer gene therapeutic molecule,the effects on leukemia remain elusive.In our protophase study,we find mda-7/IL-24 and its' alternative splice variant mda-7/IL-24asl to be present in induced U937,HL60 cells,and levels appear to increase with differentiation degree.But so far the role of mda-7/IL-24asl and mda-7/IL-24asl remain unknow.We have therefore undertaken a study to better define the significance of mda-7/IL-24 and mda-7/IL-24asl in this disease.We investigated the anti-tumor effects of ectopic expressed mda-7/IL-24 and mda-7/IL-24asl using electroporation transfection on U937 and HL60 cell lines in vitro.In 48,72 h after transfection,the expression of mda-7/IL-24,IL-24asl and IL-24-IL-24asl were ascending,and the peak time was 72h.Significant decrease of tumor cell viability was observed in U937 and HL60 cells transient transfected with mda-7/IL-24,IL-24asl,IL-24-IL-24asl compared with control cells transfected with pIRES.Transfection with mda-7/IL-24,IL-24asl and IL-24-IL-24asl also inhibited colony formation in vitro in U937 and HL60 cell lines.However,IL-24asl showed the most potent antitumor activity.Treatment of tumor cells with mda-7/IL-24, IL-24asl and IL-24-IL-24asl also resulted in a significant increase in Annexin v-positive cells,compared with plRES-transfected cells,and IL-24 had more effective on apoptosis induction.Our results show that ex vivo mda-7/IL-24,IL-24asl and IL-24-IL-24asl can induce apoptosis and growth inhibition of tumor cells.These data led us to evaluate the effect of IL-24,IL-24asl,IL-24-IL-24asl on xenograft formation by U937 and HL60 cells in nude mice.In vivo,the same antitumor activity of IL-24,IL-24asl,IL-24-IL-24asl was found.Whereafter we investigated whether ectopic expressed mda-7/IL-24,IL-24asl and IL-24-IL-24asl can induce human leukemic cells U937 and HL60 differentiation.Using FACS and morphology analysis,we found that the expression of CD11b,CD14,M-CSFR in surface of cells were significant heighten in mda-7/IL-24,IL-24asl and IL-24-IL-24asl-transfected cells,and we found that IL-24asl had more apparent effect in monocytic cell differentiation.Some strong evidence of monocyte-macrophage differentiation in morphology were found in these cells, including a decrease of the nucleus-to-cytoplasm ratio,a decrease of basophilic cytoplasmic appearance,an increase of overall cell size,appearance of pykno-chromatinic,and the induction of cytoplasmic vesicles.Although IL-24 and IL-24asl were present in differentiated monocytic cells induced by TPA,ectopic expressed IL-24 and mda-7/IL-24asl were not more effective than either of them. This maybe due to there was no synergetic effect between them.Taken together,these data strongly support that mda-7/IL-24,IL-24asl and IL-24-IL-24asl play a differentiated role in monocytic cells,they may be an effective candidate in hematopoietic malignances gene therapy.In order to prove above phenomena,U937 and HL60 cell lines were transfected with IL-24 and IL-24asl-targeted siRNA or negative control siRNA by using electroporator,at the same time TPA was added into the each system to induce the expression of IL-24 and IL-24asl.IL-24 and IL-24asl-targeted siRAN specifically reduced IL-24 and IL-24asl expression by 80%as compared with control groups.The level ofCD11b,CD14,M-CSFR were detected by FACS showed that CD11b,CD14,M-CSFR were apparently lower in siRNA-transfected cell lines than negative control siRNA- transfected cell lines and nontransfected group.In morphology, siRNA-transfected cell lines were more immature than negative control siRNA-transfected cell lines and nontransfected group.On the whole,IL-24 and IL-24asl-targeted siRNA can reduce these genes expression,and reduce the differentiated role of TPA in U937 and HL60 cell lines,therefore,IL-24,IL-24asl were relevant with monocytic differentiation.In this research we also describe our data from patients who were M5 acute monoblastic leukemia.These BMMC were transfected by electroporation,the results demonstrated that mda-7/IL-24,IL-24asl and IL-24-1L-24asl can induce a higher expression of CD11b,CD14 or M-CSFR in different degree than control.And IL-24asl showed more significant role in these genes.In the same time these genes produced a higher ratio of apoptosis than control group in two of these patients, moreover they can induce the monocyte-macrophage differentiation in morphology. However,perhaps owing to the individual difference or heterogeneity of BMMC,conspicuous differences between IL-24,IL-24asl and IL-24-IL-24asl were not found in these tested samples.In a word,IL-24,IL-24asl and IL-24-IL-24asl can induce immature cells of M5 acute monoblastic leukemia differentiation.These intriguing observations indicate that splicing of mda-7/IL-24 and IL-24 should be important in growth inhibition in hematopoietic malignances cell lines and in monocyte-macrophage differentiation and deserve further investigation.This may be an effective candidate in M5 acute monoblastic leukemia gene therapy.