Fast apoptosis and erythroid differentiation induced by imatinib mesylate in JURL-MK1 cells

We compare the effects of Imatinib mesylate (Glivec) on chronic myeloid leukemia derived cell lines K562 and JURL-MK1. In both cell lines, the cell cycle arrests in G(1)/G(0) phase within 24 h after the addition of 1 microM Imatinib. This is followed by a decrease of Ki-67 expression and the induction of apoptosis. In JURL-MK1 cells, the apoptosis is faster in comparison with K562 cells: the caspase-3 activity reaches the peak value (20 to 30 fold of the control) after about 40 h and the apoptosis proceeds to its culmination point, the DNA fragmentation, within 48 h following 1 microM Imatinib addition. Unlike K562 cells, JURL-MK1 cells possess a probably functional p53 protein inducible by TPA (tetradecanoyl phorbol acetate) or UV-B irradiation. However, no increase in p53 expression was observed in Imatinib-treated JURL-MK1 cells indicating that the difference in the apoptosis rate between the two cell lines is not due to the lack of p53 in K562 cells. Imatinib also triggers erythroid differentiation both in JURL-MK1 and K562 cells. Glycophorin A expression occurred simultaneously with the apoptosis, even at the single cell level. In K562 cells, but not in JURL-MK1 cells, the differentiation process involved increased hemoglobin synthesis. However, during spontaneous evolution of JURL-MK1 cells in culture, the effects produced by Imatinib progressively changed from the fast apoptosis to the more complete erythroid differentiation. We suggest that the apoptosis and the erythroid differentiation are parallel effects of Imatinib and their relative contributions, kinetics and completeness are related to the differentiation stage of the treated cells.     

Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.     

Drosophila p53 is a structural and functional homolog of the tumor suppressor p53

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.     

Histone deacetylase inhibitors differentially stabilize acetylated p53 and induce cell cycle arrest or apoptosis in prostate cancer cells

In LNCaP prostate cancer cells CG-1521, a new inhibitor of histone deacetylases, alters the acetylation of p53 in a site-specific manner. While p53 is constitutively acetylated at Lys320 in LNCaP cells, treatment with CG-1521, stabilizes the acetylation of p53 at Lys373, elevating p21 (and inducing cell cycle arrest). Treatment with CG-1521 also promotes Bax translocation to the mitochondria and cleavage, and apoptosis. TSA stabilizes the acetylation of p53 at Lys382, elevating p21 levels and inducing cell cycle arrest, but does not induce Bax translocation or apoptosis. In LNCaP cells CG-1521, but not TSA, promotes the rapid degradation of HDAC2. These data suggest that the acetylation of p53 at Lys373 is required for the p53-mediated induction of cell cycle arrest and apoptosis, while acetylation of p53 at Lys382 induces only cell cycle arrest. In p53(-/-) PC3 cells both compounds induce p21 and cell cycle arrest, but not Bax translocation or apoptosis, suggesting that both compounds can also induce p21 through a p53-independent mechanism.     

Inducible p27(Kip1) expression inhibits proliferation of K562 cells and protects against apoptosis induction by proteasome inhibitors

Overexpression of the cyclin-dependent kinase inhibitor p27(Kip1) has been demonstrated to induce cell cycle arrest and apoptosis in various cancer cell lines, but has also been associated with the opposite effect of enhanced survival of tumor cells and increased resistance towards chemotherapeutic treatment. To address the question of how p27(Kip1) expression is related to apoptosis induction, we studied doxycycline-regulated p27(Kip1) expression in K562 erythroleukemia cells. p27(Kip1) expression effectively retards proliferation, but it is not sufficient to induce apoptosis in K562 cells. p27(Kip1)-expressing K562 cells, however, become resistant to apoptosis induction by the proteasome inhibitors PSI, MG132 and epoxomicin, in contrast to wild-type K562 cells that are efficiently killed. Cell cycle arrest in the S phase by aphidicolin, which is not associated with an accumulation of p27(Kip1) protein, did not protect K562 cells against the cytotoxic effect of the proteasome inhibitor PSI. The expression levels of p27(Kip1) thus constitute an important parameter, which decides on the overall sensitivity of cells against the cytotoxic effect of proteasome inhibitors.     

Influence of culture medium pH on cytotoxicity of CD95L in vitro

CD95L belongs to the tumor necrosis factor-alpha (TNF-alpha) family, the members of which induce apoptosis by activation of their specific receptors. However, there are a few publications suggesting that two of these factors, TNF-alpha and TNF-beta, are able to reveal cytotoxic effect in pH-dependent manner. Therefore we investigated, whether CD95L may also reveal pH-dependent cytotoxicity. We analyzed influence of CD95L on U937 and K562 human cell lines at pH 5.1 and pH 7.4 using radioactive chromium release and tetrazolium salt (MTT) reduction assays. Expression of CD95 in both cell lines was estimated using RNase Protection Assay and FACS analysis. It has been found that short incubation of cells at pH 5.1 did not visibly affect their viability, as measured after 16 or 20 h. Incubation of U937 with CD95L at pH 7.4 resulted in a dose-dependent cell cytotoxicity. The effect was significantly augmented by incubation of cells with CD95L at pH 5.1. K562 cell line was resistant to CD95L at pH 7.4. This result correlated with the lack of CD95 expression in K562 cells. However, incubation at pH 5.1 resulted in a sensitization of K562 cells to CD95L. Our results suggest that CD95L, similarly to TNF-alpha, is able to reveal its cytotoxic activity in a receptor-independent manner and this activity strongly depends on pH of the environment.     

MYC accelerates p21CIP‐induced megakaryocytic differentiation involving early mitosis arrest in leukemia cells

p21^CIP is a potent cell cycle inhibitor often up‐regulated in differentiation. Protooncogene MYC induces cell growth and proliferation, inhibits differentiation and represses p21^CIP. However, both molecules are involved in processes of polyploidisation, cell size increase, differentiation and senescence. It is unclear why MYC has a dual role in differentiation. We have previously shown that overexpression of p21^CIP in K562 myeloid cells induces megakaryocytic differentiation with polyploidy. We have now investigated the requirements for p21^CIP to block mitosis and induce differentiation in the presence of overactivated MYC. Silencing and over‐expression studies showed that p21^CIP is required to induce differentiation. However, the expression of p21^CIP needs to be transient to irreversibly inhibit mitosis but not DNA replication, what leads to polyploidy. Transient overexpression of p21^CIP caused early down‐regulation of mitotic Cyclins and up‐regulation of G1/S Cyclins D and E, changes typical of endoreplication. Interestingly, over‐activation of MYC did not release the proliferative block imposed by p21^CIP and instead, accelerated cell size increase, megakaryocytic differentiation and polyploidisation. Our data suggests that in some systems p21^CIP takes part in a mitosis control driving MYC‐induced cellular growth into differentiation. J. Cell. Physiol. 227: 2069–2078, 2012. © 2011 Wiley Periodicals, Inc.     

All-trans-retinoic acid-mediated modulation of p53 during neural differentiation in murine embryonic stem cells

All-trans-retinoic acid (RA) plays an important physiological role in embryonic development and is teratogenic in large doses in almost all species. p53, a tumor suppressor gene encodes phosphoproteins, which regulate cellular proliferation, differentiation, and apoptosis. Temporal modulation of p53 by retinoic acid was investigated in murine embryonic stem cells during differentiation and apoptosis. Undifferentiated embryonic stem cells express a high level of p53 mRNA and protein followed by a decrease in p53 levels as differentiation proceeds. The addition of retinoic acid during 8–10 days of differentiation increased the levels of p53 mRNA and protein, accompanied by accelerated neural differentiation and apoptosis. Marked increase in apoptosis was observed at 10–20 h after retinoic acid treatment when compared with untreated controls. Retinoic acid-induced morphological differentiation resulted in predominantly neural-type cells. Maximum increase in p53 mRNA in retinoic acid-treated cells occurred on day 17, whereas maximum protein synthesis occurred on days 14–17, which coincided with increased neural differentiation and proliferation. Increased p53 levels did not induce p21 transactivation, interestingly a decrease in p21 was observed on day 17 on exposure to retinoic acid. The level of p53 declined by day 21 of differentiation. The results demonstrated that retinoic acid-mediated apoptosis preceded the changes in p53 expression, suggesting that p53 induction does not initiate retinoic acid-induced apoptosis during development. However, retinoic acid accelerated neural differentiation and increased the expression of p53 in proliferating neural cells, corroborated by decreased p21 levels, indicating the importance of cell type and stage specificity of p53 function. This revised version was published online in July 2006 with corrections to the Cover Date.     

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.     

PIPKIIα is widely expressed in hematopoietic-derived cells and may play a role in the expression of alpha- and gamma-globins in K562 cells

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.     

p21 promotes ceramide-induced apoptosis and antagonizes the antideath effect of Bcl-2 in human hepatocarcinoma cells

p21, a potent cyclin-dependent kinase inhibitor, has been known to induce cell cycle arrest in response to DNA-damaging agents. Although p21 has been reported to play an important role in the regulation of apoptosis, the postulated role for p21 in apoptosis is still controversial. Previously, we reported that p21 was induced in a p53-independent manner during ceramide-induced apoptosis in human hepatocarcinoma cell lines. In the present study, we investigated the precise role of p21 in ceramide-induced apoptosis in human hepatocarcinoma cells by using a tetracycline-inducible expression system. Overexpression of p21 by itself did not induce apoptosis in p53-deficient Hep3B cells. However, Hep3B/p21 cells were more sensitive to ceramide-induced apoptosis. In these cells, p21 overexpression did not result in G1 arrest. The expression level of Bax was increased in Hep3B/p21 cells treated with ceramide and its expression was more accelerated under the p21-overexpressed condition compared to that of the p21-repressed condition. Overexpression of Bax induced apoptosis in Hep3B cells. On the other hand, the levels of p21 and Bax protein were increased by ceramide in another hepatocarcinoma cell line, SK-Hep-1, while the Bcl-2 protein level was not changed. Overexpression of Bcl-2 not only suppressed apoptosis but also completely prevented induction of p21 and Bax caused by ceramide in SK-Hep-1 cells. Furthermore, overexpression of p21 antagonized the death-protective function of Bcl-2 and upregulated expression of Bax protein. These results suggest that p21 promotes ceramide-induced apoptosis by enhancing the expression of Bax, thereby modulating the molecular ratio of Bcl-2:Bax in human hepatocarcinoma cells.     

胡桃楸提取液诱导K562细胞凋亡机制的研究

目的：考察胡桃楸提取液是否具有诱导K562细胞p52蛋白及p21蛋白的作用,初步探讨胡桃楸提取液诱导K562细胞凋亡机制。方法：以Western-blot方法检测胡桃楸提取液对K562细胞p53蛋白及诱导K562细胞p53蛋白和p21蛋白的表达。     

Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes

HLA-G is involved in regulating T cell responses. Various mechanisms have been proposed to explain the inhibition of T cell proliferation. In this context, the possible role of HLA-G in cell cycle regulation remains to be explored. Using stably transfected M8 cells expressing the secreted isoform (HLA-G5) of HLA-G, we investigated the role of HLA-G in inducing apoptosis and in controlling the cell cycle of activated T cells. Soluble HLA-G (HLA-G5) inhibited both CD4 and CD8 T cell proliferation. However, HLA-G5 did not induce T cell apoptosis, as determined by 3,3'-diethyloxacarbocyanine and propidium iodine labeling. It induced accumulation of the retinoblastoma protein, but not its phosphorylated and active form. Treatment of activated T cells with HLA-G5 also reduced the amounts of cyclin D2, E, A, and B by >80%. In contrast, it induced an accumulation of p27kip, but not p21cip, in activated T cells. HLA-G does not induce apoptosis of alloreactive T cells, but induces p27kip1 and inhibits cell cycle progression.     

铁剥夺在TPA 诱导K562 细胞分化中对细胞 生长分化及EGR1mRNA 表达的影

目的：通过,IPA诱导K562细胞分化过程中干预细胞铁代谢探讨白血病细胞铁与细胞分化的关系及对EGR1mRNA表达的影响。方法：应用体外细胞培养技术通过细胞形态,细胞化学染色观察细胞生长分化情况;用FCM、RT—PCR等技术检细胞周期、细胞表面分化抗原CD33、CD14及EGR1mRNA的表达。结果：在,IPA诱导K562细胞分化过程中铁剥夺可明显抑制K562细胞生长,并可阻止,IPA诱导K562细胞分化,使K562细胞停止在S期。铁剥夺可降低,TPA诱导K562细胞分化过程中EGR1mRNA的表达。讨论：铁剥夺明显抑制K562细胞生长、阻止TPA诱导K562细胞分化,故铁剥夺剂（DFO）可能作为一种辅助抗癌药用于白血病的化疗,但由于它能阻止白血病细胞的分化,故不宜用于白血病的诱导分化治疗。铁剥夺使K562细胞分化过程中E—GR1mRNA表达降低可能参与了阻止TPA诱导K562细胞的分化过程。