Ribose enhances retinoic acid-induced differentiation of HL-60 cells.

Ribose, a critical building block for nucleotides, plays an important role in energy metabolism, transcription, translation, and second messenger systems. This 5-carbon sugar, synthesized from glucose via the pentose phosphate pathway, has a rate-limiting step at glucose-6-phosphate dehydrogenase. Therefore, we hypothesized that when cells are required to proliferate or differentiate, as in an immune response, the requirement for D-ribose may be greater than what could be supplied by the synthetic pathway. We hypothesized that providing an exogenous source of D-ribose during cell differentiation will enhance the process of differentiation. We used a retinoic acid-induced HL-60 cell differentiation culture as a model of neutrophil maturation. The addition of 10 to 25 mmol/L D-ribose was shown to reduce cell proliferation and move the cell population toward apoptosis in a dose-dependent manner. The expression of a cell surface marker representing maturity (CD11b) significantly increased and a cell surface marker indicative of immaturity (CD117) significantly decreased. Functionally, the cells had a greater oxidative burst function dependent on time and dose. The mechanism by which ribose enhances HL-60 cell differentiation is not known; however, as adenosine triphosphate levels did not change, adenosine triphosphate is not thought to be involved. We conclude that in this cell culture model, ribose supplementation enhanced cellular differentiation and function. Thus, ribose might be conditionally essential during time of higher need as in an immune response.

Immunostaining evidence for PI(4,5)P2 localization at the leading edge of chemoattractant-stimulated HL-60 cells.

It is well known that in fMLP-stimulated neutrophils, phosphatidyl inositol 3,4,5-trisphosphate [PI(3,4,5)P3] localizes at the leading edge of the cells. However, no effort has been made to study the PI 4,5-bisphosphate [PI(4,5)P2] distribution in these cells. In fact, it has been suggested that PI(4,5)P2 is unlikely to localize, as its basal level is orders of magnitude higher than that of PI(3,4,5)P3. We developed an optimized immunostaining protocol for studying the endogenous distribution of PI(4,5)P2 in neutrophil-like HL-60 cells. We show that PI(4,5)P2 localizes sharply at the leading edge with an intensity gradient similar to that for PI(3,4,5)P3. The enzymes for the production of PI(4,5)P2, namely, PI5KIalpha and PI5KIgamma, were also found to localize at the leading edge, further supporting our finding that PI(4,5)P2 localizes at the leading edge. These results imply that complementary regulation of PI3K and phosphate and tensin homolog (PTEN) is not the sole or dominant mechanism of PI(3,4,5)P3 polarization in HL-60 cells.

Induction of cell cycle changes and modulation of apoptogenic/anti-apoptotic and extracellular signaling regulatory protein expression by water extracts of I'm-Yunity (PSP).

BACKGROUND: I'm-Yunity (PSP) is a mushroom extract derived from deep-layer cultivated mycelia of the patented Cov-1 strain of Coriolus versicolor (CV), which contains as its main bioactive ingredient a family of polysaccharo-peptide with heterogeneous charge properties and molecular sizes. I'm-Yunity (PSP) is used as a dietary supplement by cancer patients and by individuals diagnosed with various chronic diseases. Laboratory studies have shown that I'm-Yunity (PSP) enhances immune functions and also modulates cellular responses to external challenges. Recently, I'm-Yunity (PSP) was also reported to exert potent anti-tumorigenic effects, evident by suppression of cell proliferation and induction of apoptosis in malignant cells. We investigate the mechanisms by which I'm-Yunity (PSP) elicits these effects. METHODS: Human leukemia HL-60 and U-937 cells were incubated with increasing doses of aqueous extracts of I'm-Yunity (PSP). Control and treated cells were harvested at various times and analyzed for changes in: (1) cell proliferation and viability, (2) cell cycle phase transition, (3) induction of apoptosis, (4) expression of cell cycle, apoptogenic/anti-apoptotic, and extracellular regulatory proteins. RESULTS: Aqueous extracts of I'm-Yunity (PSP) inhibited cell proliferation and induced apoptosis in HL-60 and U-937 cells, accompanied by a cell type-dependent disruption of the G1/S and G2/M phases of cell cycle progression. A more pronounced growth suppression was observed in treated HL-60 cells, which was correlated with time- and dose-dependent down regulation of the retinoblastoma protein Rb, diminution in the expression of anti-apoptotic proteins bcl-2 and survivin, increase in apoptogenic proteins bax and cytochrome c, and cleavage of poly(ADP-ribose) polymerase (PARP) from its native 112-kDa form to the 89-kDa truncated product. Moreover, I'm-Yunity (PSP)-treated HL-60 cells also showed a substantial decrease in p65 and to a lesser degree p50 forms of transcription factor NF-kappaB, which was accompanied by a reduction in the expression of cyclooxygenase 2 (COX2). I'm-Yunity (PSP) also elicited an increase in STAT1 (signal transducer and activator of transcription) and correspondingly, decrease in the expression of activated form of ERK (extracellular signal-regulated kinase). CONCLUSION: Aqueous extracts of I'm-Yunity (PSP) induces cell cycle arrest and alterations in the expression of apoptogenic/anti-apoptotic and extracellular signaling regulatory proteins in human leukemia cells, the net result being suppression of proliferation and increase in apoptosis. These findings may contribute to the reported clinical and overall health effects of I'm-Yunity (PSP).

Betulinic acid enhances 1alpha,25-dihydroxyvitamin D3-induced differentiation in human HL-60 promyelocytic leukemia cells.

Betulinic acid (BA) is a pentacyclic triterpene found in a number of medicinal plants and has been shown to cause apoptosis in a number of cell lines. We report here that BA may also have an effect on HL-60 cell differentiation. BA was cytotoxic to HL-60 cells with an IC50 of 5.7 microM after a 72-h treatment. Flow cytometry analysis showed that after exposure to 1-12 microM of BA for 72 h, approximately 10% of viable cells were in the sub-G1, presumably apoptotic, phase. At the same time differentiation was induced in approximately 10% (at 1 microM BA) to a maximum of 20% (at 6 microM BA) of cells as judged by the NBT-reduction test, and the expression of membrane markers CD11b and CD14. On the other hand, at 1 and 5 nM, 1alpha,25-dihydroxyvitamin D3 (DHD3) induced differentiation in approximately 10 and 70% of cells, respectively. At 1 nM DHD3, the addition of 1 microM BA increased differentiated cells from 10 to 43% and with 3 microM BA the increase was to 80%. BA also enhanced the effects of DHD3 in the expansion of the G1 cell population with a concomitant decrease of S phase cells. The effects of DHD3 and BA on CD11b and CD14 expression were inhibited by PD98059, a MEK inhibitor. Our results suggest that BA may enhance the effect of DHD3 in inducing mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase-mediated HL-60 cell differentiation.

[Effect of all trans retinoid acid (ATRA) on differentiation and apoptosis of HL-60 cell].

BACKGROUND & OBJECTIVE: Treatment of premyeloid leukemia with all trans retinoid acid (ATRA) is a milestone in the history of chemotherapy of malignant tumor. Previous studies suggested that the mechanism of treating premyeloid leukemia with ATRA is inducing premyeloid leukemia cells to differentiate along myelocyte lineage, but the fate of differentiated tumor cells is not clear. This study was designed to investigate the relationship between the differentiation of HL-60 induced by ATRA and apoptosis. METHODS: HL-60 cells influenced by ATRA (10 micromol/L) capable of inducing differentiation for different time were used as the subject. The differentiation marker on the cell surface and cell cycle were analyzed using flow cytometry. The differentiated cells were identified by confocal microscope after having been stained with propidium iodide (PI). Meanwhile,the changes of the apoptosis of the cells induced by ATRA at different time were analyzed using flow cytometry. RESULTS: (1)With drug-inducing time increasing, the volume of the differentiated cells was enlarged gradually. After 72 hours, the differentiated cells began to express differentiating marker CD11b and the nuclei morphology of the differentiated cells was changed. (2)After 96 hours of drug-inducing, the induced cells began to show apoptosis peak, but when the cells was washed once after 72 hours of drug-inducing with RPMI 1640 medium and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum and then cultured in 5%CO2, 37 centigrade for 8 hours,the cells began to show apoptosis peak,and the apoptosis peak was higher than that of the cells after 96 hours of drug-inducing. CONCLUSION: ATRA cannot induce HL-60 to achieve terminal differentiation,but the differentiation of HL-60 can be induced by ATRA and the differentiated leukemia cells are easy to apoptosis.

Alteration in the expression of early stage processing enzymes of N-glycan during myeloid and monocytoid differentiation of HL-60 cells.

The expressions of the enzymes participating in the early stage of N-glycan processing, Golgi alpha-Mase-I, alpha-Mase-II and GnT-I, GnT-II, were studied before and after HL-60 cells were differentiated to myelocytes or monocytes induced by ATRA or PMA, respectively. It was found that alpha-Mase-I activity and GnT-I mRNA were decreased by both ATRA and PMA, while alpha-Mase-II and GnT-II were altered insignificantly. The down-regulation of alpha-Mase-I and GnT-I was cell specific, since ATRA up-regulated alpha-Mase-I and GnT-I in the H7721 hepatocarcinoma cell line. However, in H7721 cells, PMA also decreased alpha-Mase-I and GnT-I, and both ATRA and PMA also did not obviously change the expressions of alpha-Mase-II and GnT-II.

Taraxinic acid, a hydrolysate of sesquiterpene lactone glycoside from the Taraxacum coreanum NAKAI, induces the differentiation of human acute promyelocytic leukemia HL-60 cells.

The present work was performed to elucidate the active moiety of a sesquiterpene lactone, taraxinic acid-1'-O-beta-D-glucopyranoside (1). from Taraxacum coreanum NAKAI on the cytotoxicity of various cancer cells. Based on enzymatic hydrolysis and MTT assay, the active moiety should be attributed to the aglycone taraxinic acid (1a). rather than the glycoside (1). Taraxinic acid exhibited potent antiproliferative activity against human leukemia-derived HL-60. In addition, this compound was found to be a potent inducer of HL-60 cell differentiation as assessed by a nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD 14 and CD 66 b surface antigens. These results suggest that taraxinic acid induces the differentiation of human leukemia cells to monocyte/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during taraxinic acid-dependent HL-60 cell differentiation, whereas p21(CIP1) and p27(KIP1) were up-regulated. Taken together, our results suggest that taraxinic acid may have potential as a therapeutic agent in human leukemia.

Regulation of cell cycle progression and apoptosis by beta-carotene in undifferentiated and differentiated HL-60 leukemia cells: possible involvement of a redox mechanism.

Although epidemiologic studies have demonstrated that a high intake of vegetables containing beta-carotene lowers the risk of cancer, recent intervention studies have revealed that beta-carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that beta-carotene may act as a pro- or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide-differentiated HL-60 cells exposed to beta-carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose-dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, beta-carotene was more effective in decreasing cyclin A and Bcl-2 expression and in increasing p21 and p27 expression. Neither Bcl-xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by beta-carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, alpha-tocopherol minimized the effects of beta-carotene on cell growth. These data provide evidence that beta-carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells.

Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART.

Prostratin is a unique phorbol ester that stimulates protein kinase C activity but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo human immunodeficiency virus type 1 (HIV-1) infection yet up-regulate viral expression from latent proviruses. Prostratin's lack of tumor promotion, coupled with its ability to block viral spread yet induce latent proviral expression, prompted studies to determine whether this compound could serve as an inductive adjuvant therapy for patients treated with highly active antiretroviral therapy (HAART). The current experiments indicate that prostratin is a potent mitogen for mononuclear phagocytes possessing many of the activities of phorbol myristate acetate (PMA) with notable functional differences. Prostratin, like PMA, accelerates differentiation of the myeloid cell-lines, HL-60 and THP-1, as well as mononuclear phagocytes from bone marrow and peripheral blood. Enzyme-linked immunosorbent assay and gene array analyses indicate significant changes in the expression of proteins and messenger RNA after treatment of cells with prostratin, consistent with phagocyte activation and differentiation. Prostratin blocks HIV-1 infection relating to down-regulation of CD4 receptor expression. The array analysis indicates a similar down-regulation of the HIV-1 coreceptors, CXCR4 and CCR5, and this may also reduce viral infectivity of treated host cells. Finally, prostratin is capable of up-regulating HIV-1 expression from CD8+ T lymphocyte-depleted peripheral blood mononuclear cells of patients undergoing HAART. This novel observation suggests the agent may be an excellent candidate to augment HAART by inducing expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in certain individuals infected with HIV-1.

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, induces apoptosis in human promyelocytic leukemia cells.

Extracts of the whole herb of Artemisia asiatica Nakai (Asteraceae) have been used in traditional oriental medicine for the treatment of inflammation, cancer and other disorders. In the present work, we have evaluated the apoptosis-inducing capability of eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone), a pharmacologically active ingredient of A. asiatica, in cultured human promyelocytic leukemia (HL-60) cells. Thus, eupatilin exhibited concentration-dependent inhibitory effects on viability and DNA synthesis capability of HL-60 cells. The anti-proliferative effect of eupatilin was attributable to its apoptosis-inducing activity as determined by characteristic nuclear condensation, in situ terminal end-labeling of fragmented DNA (TUNEL), release of mitochondrial cytochrome c into cytoplasm, proteolytic activation of caspases-9, -3, and -7, and cleavage of poly(ADP-ribose)polymerase. Eupatilin-induced HL-60 cell apoptosis does not appear to be mediated via alteration in Bcl-2/Bax-2. Taken together, the above findings suggest that eupatilin has chemopreventive and cytotoxic effects.

[Preliminary study on apoptosis of human leukemic cells induced by bufalin].

OBJECTIVE: To study the mechanism of inhibiting actions of bufalin on human leukemic cells (HL60). METHODS: HL60 cells were incubated with bufalin at different concentrations and growth inhibition was analyzed by trypan-blue staining. Cell apoptosis was evaluated by phase contrast microscopy, transmission eletron microscopy and agarose gel electrophoresis. RESULTS: Bufalin remarkably inhibited the growth of HL60 cells. Apoptosis of HL60 cells could be effectively induced by bufalin at concentration of 0.01 mumol.L-1 or higher, showing the apoptotic changes in morphology, including cell shrinkage, chromatin agglutination and formation of apoptotic corpuscula; DNA cleavage was observed with agarose gel electrophoresis. CONCLUSION: Bufalin can effectively induce apoptosis of HL60 cells, which is possibly one of the mechanisms for anti-cancer of bufalin.

Induction of apoptosis by hydrolyzable tannins from Eugenia jambos L. on human leukemia cells.

Eugenia jambos L. (Myrtaceae) is an antipyretic and anti-inflammatory herb of Asian folk medicine. A 70% acetone extract exerted the strongest cytotoxic effects on human leukemia cells (HL-60) from a preliminary screening of 15 plants. The cytotoxic principles were separated by bio-assay-guided fractionation to HL-60 cells; two hydrolyzable tannins (1-O-galloyl castalagin and casuarinin) were isolated from the 70% acetone extract. All significantly inhibited human promyelocytic leukemia cell line HL-60 and showed less cytotoxicity to human adenocarcinoma cell line SK-HEP-1 and normal cell lines of human lymphocytes and Chang liver cells. Thus, these compounds were exhibited the dose-dependent manner in HL-60 cells and the IC(50) were 10.8 and 12.5 microM, respectively. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G(2)/M phase, and a concomitant increase of cell population at G(1) phase. The apoptosis induced by these two compounds was also demonstrated by DNA fragmentation assay and microscopic observation. These results suggest that the cytotoxic mechanism of both antitumor principle constituents might be the induction of apoptosis in HL-60 cells.

Alterations of the actin polymerization status as an apoptotic morphological effector in HL-60 cells.

The alterations of the cytoskeletal actin network have been implicated as a morphological effector in apoptosis. However, studies directly linking actin change to the morphological events in apoptosis are lacking. This study quantitatively examined the effect of actin alteration on the camptothecin (CPT)-induced apoptotic process in HL-60 cells. Actin alteration was induced by two distinctive types of agent: the polymerization-stimulating agent, Jasplakinolide (Jas), and the polymerization-blocking agent, cytochalasin B (CB). The actin polymerization status was measured by two complementary methods: the cell pellet-based DNase I inhibition method, and the individual cell-based quantitative fluorescence image analysis (QFIA) assay. Actin polymerization induced by Jas caused apoptosis directly. By contrast, CB, an actin polymerization-blocking agent, partially inhibited CPT-induced apoptosis. A similar inhibition of the CPT-induced apoptosis response was observed with a more specific actin depolymerization agent, cytochalasin E. The alterations of the actin polymerization status occurred in three sequential steps during the apoptotic process: first polymerization, followed by depolymerization, and finally degradation. However, compared with CPT-induced apoptosis, Jas-induced apoptosis was characterized by pronounced actin polymerization that corresponded morphologically with prominent membrane blebbing, but less apoptotic body formation. Furthermore, DNase I activity, which is normally inhibited by G-actin, was specifically detected in Jas-treated cells. These results show that the regulation of actin polymerization is an important apoptotic morphological effector, whereas the alterations of the actin polymerization status by chemicals have profound effects not only on altering the morphology of apoptotic cells, but on apoptosis induction in HL-60 cells as well.

Steroidal saponins from the rhizomes of Hosta sieboldii and their cytostatic activity on HL-60 cells.

A total of eighteen steroidal saponins were isolated from the rhizomes of Hosta sieboldii, one of which appeared to be the first isolation from a plant source and six to be new compounds. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (25R)-2 alpha, 3 beta-dihydroxy-5 alpha-spirostan-12-one (manogenin) 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D-glucopyranosyl -(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha,3 beta-dihydroxy-5 alpha-spirost-9-en-12-one (9,11-dehydromanogenin) 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, 9,11-dehydromanogenin 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[O-alpha-L- rhamnopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->3)]-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha,3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furostan-12-one 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha, 3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furost-9-en-12-one 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿ and (25R)-5 alpha-spirostan-2 alpha,3 beta,12 beta-triol 3-O-¿O-alpha-L- rhamnopyranosyl-(1-->2)-beta-D-galactopyranoside¿, respectively. Cytostatic activity of the isolated saponins on leukaemia HL-60 cells was examined.

Cloning of human ENC-1 and evaluation of its expression and regulation in nervous system tumors.

We recently identified and characterized a novel murine gene, ENC-1, that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role for ENC-1 gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue of ENC-1. The human ENC-1 gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels of ENC-1 mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of the ENC-1 gene during neural crest cell differentiation. We found that the expression of ENC-1 increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest that ENC-1 expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.

Increasing c-FMS (CSF-1 receptor) expression decreases retinoic acid concentration needed to cause cell differentiation and retinoblastoma protein hypophosphorylation.

Increasing the expression of c-FMS (colony-stimulating factor 1 receptor) by introduction of a transgene reduced the concentration of retinoic acid or 1,25-dihydroxy vitamin D3 needed to cause myeloid or monocytic cell differentiation and hypophosphorylation of the retinoblastoma tumor suppressor protein (RB) typically associated with cell cycle G0 arrest and differentiation of HL-60 human myelo-monoblastic precursor cells. The data are consistent with a model in which signals originating with retinoic acid and c-FMS integrate to cause differentiation, RB hypophosphorylation, and G0 arrest. Furthermore, these two signals can compensate for each other. Three HL-60 sublines described previously (A. Yen et al., Exp. Cell Res., 229: 111-125, 1996) expressing low (wild-type HL-60), intermediate, and high cell surface c-FMS were treated with various concentrations of retinoic acid. The lowest concentration tested, 10(-8) M, induced significant differentiation of only the high c-FMS-expressing cells, with no accompanying hypophosphorylated RB or G0 arrest. The low and intermediate c-FMS expressing cells showed no induced differentiation, hypophosphorylation of RB, or G0 arrest. A 10-fold higher retinoic acid concentration, 10(-1) M, induced significant differentiation of both intermediate and high c-FMS-expressing cells. It induced RB hypophosphorylation only in high c-FMS-expressing cells but with no accompanying G0 arrest in any of the cells. The highest retinoic acid concentration, 10(-6) M, elicited differentiation, hypophosphorylation of RB, and G0 arrest in low, intermediate, and high c-FMS-expressing cells. As the concentration of retinoic acid increased, cell differentiation, hypophosphorylation of RB, and G0 arrest were progressively elicited within this ensemble of cells with different c-FMS expression levels. Thus, for example, at the lowest concentration of retinoic acid, expression of high enough c-FMS still allowed differentiation. At higher concentrations, progressively less c-FMS was needed for differentiation. The apparent threshold for the sum of the retinoic acid plus c-FMS originated signals to elicit differentiation, hypophosphorylation of RB, and G0 arrest increased, in that order. Thus retinoic acid-induced cell differentiation, RB hypophosphorylation, and G0 arrest have different signal threshold requirements. 1,25-Dihydroxy vitamin D3, also a ligand for a member of the steroid thyroid hormone receptor superfamily, caused monocytic differentiation with a similar c-FMS dependency, indicating that these effects characterize both myeloid and monocytic differentiation.

Enforced expression of Bcl-XS induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis K562 cells to 1-beta-D-arabinofuranosylcytosine-mediated differentiation and apoptosis.

Human chronic myelogenous leukemia-blast crisis K562 cells have been demonstrated to be relatively resistant to antileukemic drug-induced apoptosis. This has been attributed to the activity of p210bcr-abl tyrosine kinase present in the K562 cells, which is known to suppress drug-induced apoptosis. Recently, K562 cells have been shown to express the antiapoptosis Bcl-xL but not Bcl-2 proteins. To investigate the contribution of Bcl-xL toward resistance to drug-induced apoptosis, we created K562/Bcl-xS and K562/neo cells by electroporating the expression plasmids pSFFVneo-Bcl-xS and pSFFVneo, containing the bcl-xS and neomycin resistance genes, respectively, into K562 cells. K562/Bcl-xS but not K562/neo cells expressed the bcl-xS mRNA and p19Bcl-xS protein. In contrast, both cell types expressed equivalent levels of Bcl-xL, Bax, Bcl-2, Myc, retinoblastoma, p21cbor-abl, and p145abl proteins. A significant increase in the hemoglobin levels was observed in the K562/Bcl-xS compared with the K562/neo cells (P < 0.05). In addition, K562/Bcl-xS cells were significantly more sensitive than K562/neo cells to undergoing erythroid differentiation induced by low-dose 1-beta-D-arabinofuranosylcytosine (ara-C) and hexamethyl bisacetamide (P < 0.05), but not by all-trans-retinoic acid. Low-dose ara-C- or hexamethyl bisacetamide-induced differentiation was not associated with apoptosis of K562/Bcl-xS or K562/neo cells. Low-dose ara-C-induced erythroid differentiation was accompanied by conversion of the retinoblastoma protein to predominantly its underphosphorylated isoform as well as by down-regulation of Myc levels in K562/Bcl-xS and K562/neo cells. Importantly, exposure to high-dose ara-C (HIDAC; 100 microM ara-C for 4 h) caused internucleosomal DNA fragmentation and the morphological features of apoptosis in K562/Bcl-xS cells. These effects were modestly enhanced by cotreatment with HIDAC plus herbimycin A. In contrast, K562/neo cells were completely resistant to HIDAC- and herbimycin A-induced apoptosis. These results indicate that the expression of Bcl-xS induces erythroid differentiation and partially sensitizes chronic myelogenous leukemia-blast crisis-derived K562 cells to ara-C-induced differentiation and apoptosis.

The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties.

The tryptic meat digest Primatone RL is a low-cost medium supplement of a complex nature which serves as a source of amino acids, oligopeptides, iron salts, some lipids and other trace low molecular weight substances. Its addition to mammalian and insect cell culture media significantly improves the cell growth properties of many cell lines. In this work the growth promoting effects of Primatone RL are described in more detail using different mouse hybridomas, a mouse myeloma cell line, and human promyelocytic leukemia HL-60 cells. The positive effects on cell growth induced by Primatone were observed in the presence of serum but were even more pronounced in serum-free culture. In addition the adaptation time from high serum to low (1%) or serum-free growth in the presence of Primatone is also significantly reduced. Primatone RL, when added to HL and DHI medium, improves cell growth under low serum or serum-free conditions by increasing the maximum cell numbers and in particular the viability of the culture. The observed decrease in cell death (apoptosis) induction leads to a significant improvement in antibody (recombinant protein) production by increasing the volumetric yields during long-term batch culture. The so-called anti-apoptotic effects of Primatone RL for mouse hybridomas, which is concentration dependent, is not fully understood.

[The antitumor activity of elemene is associated with apoptosis].

Elemene, isolated from the Chinese medicinal herb Rhizoma Zedoariae was shown to exhibit antitumor activity in vitro and in vivo to human and murine tumor cells. This novel antineoplastic agent has been demonstrated to have substantial clinical activity against various tumors. In this paper, the mechanisms of antitumor activity of elemene are reported. The in vitro effect of elemene on the growth of leukemia cells was evaluated by MTT assay. The IC50 values of elemene for promyelocytic leukemia HL-60 cells and erythroleukemia K562 cells were found to be 27.5 micrograms/ml and 81 micrograms/ml, respectively, while IC50 for peripheral blood leukocytes (PBL) was 254.3 micrograms/ml. The inhibitory effect of elemene on proliferation of HL-60 cells was associated with cell cycle arrest from, S to G2M phase transition and with induction of apoptosis. The apoptosis of tumor cells was confirmed by DNA ladder formation on gel electrophoresis and characteristic ultrastructural alternations. These results indicate that induction of apoptosis contributes to the mechanisms for antitumor activity of elemene.

Rotenoids mediate potent cancer chemopreventive activity through transcriptional regulation of ornithine decarboxylase.

For the discovery of new cancer chemopreventive agents, we have studied the potential of plant extracts to inhibit phorbol ester-induced ornithine decarboxylase (ODC) activity in cell culture. Four active rotenoids were obtained from the African plant Mundulea sericea (Leguminosae). These isolates were highly potent when evaluated for inhibition of chemically induced preneoplastic lesions in mammary organ culture and inhibition of papillomas in the two-stage mouse skin model, and they appear to function by a unique mechanism at the level of ODC messenger RNA expression. Based on our findings, rotenoids can be regarded as promising new chemopreventive or anticancer agents.