| ObjectiveThe WT1 gene, which is located at the human chromosome region 11 p13, contains 10 exons. As a result of two alternative mRNA splicing, four major splicing isoforms are generated. Alternative splice I inserts or removes 17 amino acids (17aa) of exon 5, which is located in the transcriptional regulatory domain, and alternative splice II results in the insertion of three amino acids (KTS) between the third and fourth zinc -fingers. The presence or absence of exon 5 modulates the transcriptional regulatory activity, while the KTS insert affects DNA binding specificity and subcellular localization. The four major isoforms of WT1, designated WT1 ( + 17aa / + KTS) , WT1 ( + 17aa / - KTS) , WT1 ( - 17aa / + KTS) , and WT1 ( - 17aa / - KTS) to indicate the presence or absence of exon 5/KTS, respectively, are generally coexpressed in a fixed ratio in embryonic kidney and specific cell types of the adult in kidney, gonads, hematopoietic and nervous system. The isoform containing both splice inserts is the most prevalent variant, whereas the least common is the transcript missing both inserts. The + KTS/ - KTS ratio is maintained in all cell types, while the splicing of exon 5 is differentially regulated in a species - , tissue - and developmental stage - specific manner.WT1 gene, encoding a zinc finger transcription factor, was originally identified as a tumor suppressor gene for its involvement in the pathogenesis of Wilms tumor. However, WT1 was overexpressed in leukemia and a variety of solid tumors, including malignant mesothelioma, lung, breast, and renal cell cancer, and associated with poor prognosis, indicating that WT1 play a role as an onco-gene in these tumors. Recent studies has demonstrated that differentiation of leukemia cells in cultrue is accompanied by downregulation of WTl protein level, ectopic expression of WTl inhibits the differentiation and apoptosis of leukemia cells, and inhibition of WTl expression by WTl antisense oligonucleotides leads to enhanced apoptosis. These results suggested that expression of WTl might contribute to the maintenance of a malignant phenotype through a variety of mechanisms including inhibiting differentiation and apoptosis and promoting proliferation. Recently, 17aa - containing WTl transcrips were shown to be in excess in leukemias, and a high ratio of + 17aa/ - 17aa was associated with a poor prognosis. These findings indicated that the interactions between WTl four isoforms might play a important role in leukemogenesis process, but the exact mechanisms still remain poorly understood.Bufalin is one of the components of bufadienolides in the traditional Chinese medicine Chan su, which induces differentiation and apoptosis of some leukemia cells at a low or high concentration respectively. In our previous study, the total level of WTl mRNA and protein were shown to be dowmregulated in the early phase of apoptosis and differentiation of K562 cells induced by bufalin, but whether there are changes in the ratio of four isoforms still remains unclear. In present study, we examined the expression of WTl four splicing variants by RT - PCR using primers specific for KTS insert, and explored the significance of the ratio of these isoforms in apoptosis and differentiation induced by bufalin in K562 cells. Furthermore, we transfected WT1( + 17aa/ +KTS) to U937 cells, which lack detectable amounts of WTl protein, and explored the effects of WTl ( + 17aa/ + KTS) on the monocytic differentiation induced by TPA and apoptosis triggered by a variety of chemotherapeutic agent in U937 cells.Methods1. Cell cultureK562 and U937 leukemia cells were cultured in RPMI1640 supplemented with 10% ( v/v) heat - inactinvated fetal bovine serum containing 12U/ml gen-tamycin and incubated at 37^ in a 5% CO2 atmosphere. In all experiments,cells in logarithmic growth phase were used.2. Gene transfectionWT1( +17aa/ +KTS)cDNA was transfected to U937 cells by the calcium phosphate method. RT - PCR confirmed the expression of WT1 ( + 17aa/ + KTS) mRNA. The transfected U937 cells were cultured in the presence of 200p,g/ml G418, and transferred to fresh culture medium 24 hours before experiment.3. Assays for cellular proliferation and viabilityThe proliferation was determined by counting Biirker chamber and cell viability was determined by typan blue exclusion.4. Assays for differentiation1) Morphological analysis.2) NBT reduction test The percentage of cells containing formazan de-poisits, corresponding to the capacity to reduce NBT, was determined by counting 200 cells.3) Expression of the monocytic antigen CDllb was analyzed by flow cytom-etry.5. Assay for apoptosisApoptotic cells were determined by morphological analysis and flow cytome-try .6. Western blotThe expression of WT1 protein was analyzed by western blot.7. Reverse transcriptase - polymerase chain reaction (RT - PCR)The expression of WT1 four splicing variants mRNA was analyzed by RT -PCR.8. Statiscal analysisThe data were expressed as means ± SD of three independent examinations, and t - test was used to assess the significance of the difference between control and treatment groups. All data were analyzed using the software SPSS 10.0 for windows.Results1. Bufalin inhibited proliferation in K562 cellsBufalin inhibited K562 cells proliferation in a time - and dose - dependent manner at 0. OOljxmol/L ~50jxmol/L. And the IC^ of 24h, 48h and 72h were 0.19|xmol/L^0.021|xmol/L and 0.006|xmol/L respectively.2. Bufalin induced apoptosis in K562 cellsFollowing treatment with 0. 5jxmol/L ~2. Oujnol/L bufalin for 24h, K562 cells exhibited typical apoptotic features such as cell shrinkage, nuclear condensation and formation of apoptotic bodies. Flow cytometric analysis revealed a time - and dose - dependent sub - Gl peak.3. Bufalin induced differentiation inK562 cellsFollowing treatment with 0.01 u,mol/L ~ 0.1 ujmol/L bufalin for 6 days, the NBT reducing capacity of K562 cells was enhanced. And typical morphological features of mature monocytes/macrophages were observed.4. The expression of WT1 protein was downregulated by bufalin Following treatment of K562 cells with 2.0 jxmol/L bufalin, western blot a-nalysis revealed that WT1 protein was gradually reduced from 8h,completely disappeared at 24h. And following treatment with 0.05 jxmol/L bufalin, WT1 protein was decreased from Id, completely disappeared at 4d.5. The ratio of +17aato -17aa was dowmregulated by bufalin Following treatment with 2. Ojxmol/L bufalin for 4h, the expression of +17aa was reduced, the expression of - 17aa was increased, and the ratio of + 17aa to - 17aa was rapidly decreased. At 8 hours, the levels of all four isoforms were reduced. By 24 hours post - treatment, these transcripts disappeared completely. Following treatment with 0.05|xmol/L bufalin for 1 day, the expression of + 17aa was significantly reduced, the expression of - 17aa was slightly decreased, and the ratio of + 17aa to - 17aa was also rapidly decreased. By 3 days post - treatment, these transcripts disappeared completely.6. The expression of WT1 ( + 17aa/ + KTS) did not affect the growth of U937 cellsViability of the cells, as judged by typan blue exclusion, was similar for WTl - transfectant and control cells and always more than 90%. The doubling time were 19.4h ±0.7h and 18.4h ±0.3h respectively, there was no significant difference.7. The expression of WTl ( + 17 aa/ + KTS) did not block TPA - induced differentiation of U937 cellsFollowing treatment with 10 nmol/L TPA for 3 days, typical morphological features of mature monocytes/macrophages, including adherence, nuclear convolution , decrease in the ratio of nuclear/cytoplasm, appearance of fine granules and vacuoles in the cytoplasm, were observed in both WTl - transfectant and control cells. And CDllb was also similarly upregulated.8. The expression of WTl ( + 17aa/ + KTS) inhibited apoptosis of U937 cells induced by chemotherapeutic drugsFollowing treatment with 50nmol/L mitoxantrone, 1. 6|xg/ml As2 O3, 1 ujnol/L Ara - C, 1 |xg/ml Vp - 16 and 2: Ou-mol/L bufalin for 5 hours respectively , a significant decrease in apoptosis was observed in WTl transfectant compared to control U937 cells.DiscussionWTl was overexpressed in leukemia, and associated with poor prognosis, indicating that WTl play a role as an oncogene in leukemia. Recent studies has demonstrated that differentiation of leukemia cells in cultrue is accompanied by downregulation of WTl protein levels, ectopic expression of WTl inhibits the differentiation and apoptosis of leukemia cells, and inhibition of WTl expression by WTl antisense oligonucleotides leads to enhanced apoptosis. These results suggested that expression of WTl might contribute to the maintenance of a malignant phenotype through a variety of mechanisms including inhibiting differentiation and apoptosis and promoting proliferation. In the present study, the expression of WTl total protein was downregulated in the initial stage of apoptosis and differentiation induced by bufalin. These results suggested that bufalin - induced apoptosis and differentiation might be associated with the downregulation of WTlexpression, suppoting anti - apoptosis and differentiation effects of WTl gene. However, there are several conflicting reports. Smith et al showed that WTl + KTS isoforms promoted, rather than inhibited, monocytic differentiation in the murine promyelocytic leukemia cell line, Ml. The biology of WTl is complex, and several associations with other proteins including p53 and Par - 4 have been demonstrated. These associations have the capacity to alter the transcriptional properties of WTl. Their presence or absence at different developmental stages and in different cell backgrounds can presumably modify the phenotypic effects of WTl expression.As a result of two alternative mRNA splicing, WTl gene produces four major splicing isoforms. Recently, 17aa - containing WTl transcrips were shown to be in excess in leukemias, and a high ratio of + 17aa/ - 17aa was associated with a poor prognosis. Richard et al reported that only exon 5 containing isoforms of WTl overcomed a UV - induced apoptotic signal, when transfected into HEK293 cells, suggesting that exon 5 might be essential for the regulation of cellular apoptosis. Gu et al showed the levels of expression of WTl + 17aa isoforms rapidly decreased during the differentiation of NB4 cells induced by AT-RA, and this decrease was conversely related to the dynamic changes of CDllb positive cells. In the present study, the ratio of WTl + 17aa to - 17aa isoforms were rapidly decreased during the early phases of both apoptosis and differentiation, indicating that the changes of WTl splicing pattern was associated with K562 cells differentiation and apoptosis induced by bufalin.The four major isoforms of WTl are generally coexpressed in a fixed ratio in hematopoietic progenitors, and the isoform containing both splice inserts is the most prevalent variant. In this study, WTl ( + 17aa/ + KTS) cDNA was transfected to U937 cells, which was found to lack detectable amounts of WTl protein. Following incubated in the presence of lOnmol/L TPA, WTl transfectant cells displayed morphological features of mature monocytes/macrophages and a increase in the expression of monocytic differentiation marker CDllb similarly to control cells, indicating that WTl ( + 17aa/ + KTS) did not affect TPA - induced monocytic differentiatiyon of U937 cells. However, apoptosis triggered by a variety of chemotherapeutic drugs was inhibited to some extent in WTl trans-...
|
PDF Full Text Request