[Show abstract][Hide abstract] ABSTRACT: The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-kappaB) in response to stimulation with tumor necrosis factor alpha (TNF-alpha) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-kappaB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-kappaB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-kappaB (IkappaB). In vitro kinase assays showed that RKIP antagonizes the activation of the IkappaB kinase (IKK) activity elicited by TNF-alpha. RKIP physically interacted with four kinases of the NF-kappaB activation pathway, NF-kappaB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKalpha, and IKKbeta. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-kappaB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.
Molecular and Cellular Biology 12/2001; 21(21):7207-17. · 5.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-kappaB (NF-kappaB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-kappaB and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-kappaB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-kappaB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-kappaB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-kappaB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-kappaB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Stimulation of CD95 leads to oligomerization of this receptor and the recruitment of the Fas-associated death domain (FADD) and procaspase-8 to form the death-inducing signaling complex (DISC). Subsequent proteolytic activation of caspase-8 at the DISC leads to the activation of downstream caspases and execution of apoptosis. The anticancer drug 9-nitrocamptothecin (9NC) inhibits the nuclear enzyme topoisomerase I (Top1), an event followed by apoptosis of cancer cells. We investigated whether other mechanisms downstream of the DNA-Top1-9NC complexing step regulate the apoptotic ability of 9NC in DU145 cells. We demonstrate that induction of apoptosis in DU145 cells, upon exposure to 9NC, is associated with de novo expression of CD95 and CD95L, suggesting that 9NC-induced apoptosis is mediated by the CD95 system. In this line, we observed early activation of procaspase-3, -7, and -8, but not -1, -9, and -10. Moreover, 9NC treatment resulted in the dramatic down-regulation of c-FLIP(short) expression, but not that of c-FLIP(long) or FADD. Furthermore, incubation of DU145 cells with a neutralizing antibody (NOK-1) to CD95L or transient transfection of a c-FLIP(short) expression vector into DU145 cells partially abrogated 9NC-triggered apoptosis. We propose that 9NC triggers apoptosis by driving DU145 cells from a nonapoptotic status (c-FLIP(short)(high), CD95(low), CD95L(low)) toward a proapoptotic status (c-FLIP(short)(low), CD95(high), CD95L(high)). These findings indicate that in addition to a Top1-mediated effect, 9NC can additionally activate a CD95/CD95L-dependent apoptotic pathway.
Cancer Research 11/2001; 61(19):7148-54. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.
Cancer Research 04/2001; 61(5):1964-9. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this report, we demonstrate the down-regulation of telomerase activity and c-Myc and Bcl-2 expression during 9-nitrocamptothecin (9NC)-induced regression of human DU145 prostate tumors grown as xenografts in immunodeficient mice. These changes were not observed in tumors generated by DU145-derived cells resistant to 9NC. We suggest that telomerase activity, c-Myc and Bcl-2 can collectively serve as molecular diagnostic indicators of the effectiveness of 9NC during treatment of human prostate tumors.
Anticancer research 01/2000; 20(5A):2885-9. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated changes in the content and subcellular localization of the cell cycle regulators, cyclin B1 and cyclin-dependent kinase cdc2, in human prostate DU145 tumor and cultured cells treated with the anticancer drug 9-nitrocamptothecin (9NC).
Proteins of interest were identified by Western blot methodology using specific antibodies.
The cyclin B1 and cdc2 contents were dramatically elevated in biopsies of DU145 tumor regressing upon 9NC-treatment. In vitro, 9NC-induced apoptosis of DU145 cells was associated with up-regulation of expression and nuclear accumulation of cyclin B1 and cdc2. No changes were observed in cyclins A and E and the cyclin-dependent kinase cdk2 in 9NC-treated DU145 tumor and cultured cells.
9NC-induced apoptosis in DU145 cells in vivo and in vitro is associated with up-regulation of expression and nuclear localization of cyclin B1 and cdc2.
Anticancer research 01/2000; 20(6B):4477-82. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Normal human monocytes express Fas and are susceptible to Fas ligand (FasL)-induced apoptosis. Because the myeloid leukemia cell lines HL-60 and THP-1 can be differentiated into functional monocytes and macrophages, we studied their expression of Fas and Fas ligand (FasL) to determine whether there were differentiation-associated changes in these proteins. THP-1, HL-60 and HCW-2, both before and after treatment with PMA, expressed high levels of Fas ligand (FasL), but did not express Fas. The FasL expressed by THP-1 cells was functional as measured by their ability to kill Jurkat T-cells by apoptosis. The THP-1 Fas gene appears to be silent, because bacterial lipopolysaccharide (LPS) induced Fas expression in fully differentiated THP-1 cells. Our results suggest that FasL expression by leukemia cells may account in part for the pathophysiology of myeloid leukemia, and that PMA-differentiated THP-1 cells, while possessing many of the functional properties of normal macrophages, are abnormal with respect to a major apoptotic pathway.
Leukemia Research 11/1999; 23(10):865-70. · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After in-vitro exposure to 0.05 micromol/L 9-nitrocamptothecin (9NC) for periods of time longer than 5 days, 65% to 80% of the human malignant melanoma SB1B cells die by apoptosis, whereas the remaining cells are arrested at the G2-phase of the cell cycle. Upon discontinuation of exposure to 9NC the G2-arrested cells resume cell cycling or remain arrested depending on the duration of 9NC exposure. In contrast to cycling malignant cells, the cells irreversibly arrested at G2 exhibit features of normal-like cells, the melanocytes, as assessed by the appearance of dendrite-like structures; loss of proliferative activity; synthesis of the characteristic pigment, melanin; and, particularly, loss of tumorigenic ability after xenografting in immunodeficient mice. Further, the expression of the cyclin-dependent kinase inhibitor p16 is upregulated in the 9NC-treated, G2-arrested, but downregulated in density G1-arrested cells, whereas the reverse is observed in the expression of another cyclin-dependent kinase inhibitor, p21. These results suggest that malignant melanoma SB1B cells that escape 9NC-induced death by apoptosis undergo differentiation toward nonmalignant, normal-like cells.
[Show abstract][Hide abstract] ABSTRACT: Six camptothecin (CPT) alkyl esters and four 9-nitrocamptothecin (9NC) alkyl esters were assayed for ability to inhibit proliferation and induce programmed cell death (apoptosis) in human leukemia HL-60 and U-937 cells, which exhibit differential sensitivity to CPT and 9NC. In general, CPT-propionate and CPT-butyrate demonstrated activities, while the other esters were practically inactive. Similarly, 9NC-propionate and 9NC-butyrate were active, while the other 9NC esters exhibited little or no activity. The biologically active esters required metabolic conversion (i.e., de-esterification) to their parental compounds as demonstrated by the conversion of CPT-propionate to CPT in mouse liver homogenate, and the topoisomerase I-inhibition assay. In conclusion, the propionate and butyrate esters of CPT and 9NC are CPT and 9NC prodrugs, that can develop to important chemotherapeutic agents for the effective treatment of human leukemias and other malignancies.
European Journal Of Haematology 05/1999; 62(4):246-55. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have investigated whether hyperthermia (HT) treatment at 43 degrees C for 15-60 min can affect the extent of apoptosis induced in human leukemia HL-60 cells by the anticancer drug 9-nitrocamptothecin (9NC). Quantitative changes in the apoptotic (Ap) fraction in the cell cultures were monitored by flow cytometry. The results showed that (i) heating for 15 min prior to or concurrently with 9NC exposure had no effect on the Ap fraction generated by the drug alone, whereas 60 min heating resulted in an increase in the Ap fraction; and (ii) heating of the cells at 6-24 h after exposure to the drug enhanced the Ap fraction. These results indicate that appropriate scheduling of HT and 9NC treatments may lead to thermochemotherapy protocols that will result in increased 9NC-induced death of human leukemia cells.
[Show abstract][Hide abstract] ABSTRACT: In addition to being causative agents of infectious diseases in animals and humans, DNA viruses have served as models for the study of eukaryotic molecular mechanisms including replication and transcription. Studies of DNA virus functions utilizing cell-free systems and virus-infected cells in culture, in the presence of the anticancer drug camptothecin (CPT), have demonstrated that CPT is a potent inhibitor of replication, transcription and packaging of double-stranded DNA-containing adenoviruses, papovaviruses and herpesviruses, and the single- stranded DNA-containing autonomous parvoviruses. CPT inhibits viral functions by inhibiting topoisomer- ase I, a host cell enzyme required for initiation and completion of the viral functions. These findings indicate that CPT analogues could be developed for use as potent drugs against DNA viruses.
Journal of Biomedical Science 02/1999; 6(1):1-7. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatment of human promyelocytic leukemia HL-60 cells with phorbol esters ultimately induces the differentiation of these cells along the monocyte/macrophage lineage, whereas treatment with retinoic acid or DMSO induces granulocytic/neutrophillic differentiation. In this study, we demonstrate the disparate fates of HL-60 cells treated with the phorbol ester 12,13-phorbol dibutyric acid (PDBu) or DMSO. After DMSO treatment, HL-60 cells eventually died via apoptosis, whereas the viability of PDBu-treated cells was not affected during the same interval. The levels of the apoptosis effector proteins Bak and Bad were enhanced, whereas there was a slight down-regulation of the apoptosis suppressor protein Bcl-2 after treatment of the cells with PDBu and DMSO. Treatment with DMSO resulted in the elevation of the apoptosis effector Bax, whereas treatment with PDBu did not significantly alter the levels of this protein. However, treatment of HL-60 cells with PDBu induced the rapid expression of the apoptosis suppressor protein Bcl-xL, whereas the expression of this protein remained unaltered in DMSO-treated cells. The generality of this finding was confirmed by the induction of Bcl-xL in human myeloid U-937 cells, human peripheral blood monocytes exposed to phorbol ester, and mouse thioglycollate-activated and resident peritoneal macrophages. PDBu-treated HL-60 cells remained viable for 7 days and thereafter began to die via apoptosis, with a concomitant down-regulation of Bcl-xL. In conclusion, we propose that Bcl-xL expression is associated with differentiation and survival of hematopoietic cells along the monocyte/macrophage lineage.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 11/1997; 8(10):1083-9.
[Show abstract][Hide abstract] ABSTRACT: ATP acts as an intracellular energy source and an extracellular signaling molecule. We report that extracellular ATP causes apoptosis in pulmonary artery endothelial cells, as assessed by morphological changes and internucleosomal DNA degradation. We investigated the mechanism of this effect using release of tritiated soluble DNA as a marker for apoptosis. We conclude that the metabolite adenosine is responsible for the apoptotic effect of ATP, since nucleotides that can be degraded to adenosine, as well as adenosine itself, cause DNA damage, whereas nonmetabolizable ATP analogs and uridine 5'-triphosphate are inactive. Furthermore, the ecto-5'-nucleotidase inhibitor alpha, beta-methylene-ADP blocks ATP-induced DNA fragmentation. The adenosine receptor agonist 5'-N-ethylcarboxamide adenosine does not cause DNA fragmentation, and adenosine receptor antagonists do not block adenosine-induced apoptosis. However, the nucleoside transport inhibitor dipyridamole prevents extracellular ATP-induced DNA cleavage. These findings indicate that ATP- and adenosine-mediated apoptosis are mediated via intracellular events rather than through cell surface receptor(s). The adenosine metabolites inosine, hypoxanthine, and xanthine do not cause apoptosis. The adenosine analogs 3-deazaadenosine and MDL-28842, which are not metabolized and are S-adenosylhomocysteine hydrolase inhibitors, also cause DNA fragmentation. Therefore, we speculate that extracellular ATP and adenosine cause apoptosis of pulmonary artery endothelial cells by altering methylation reactions that require S-adenosylmethionine as the methyl donor. We speculate that ATP released from cells undergoing cytolysis or degranulation may cause endothelial cell death. Endothelial cell apoptosis may be important in acute vascular injury or in limiting angiogenesis.
The American journal of physiology 09/1997; 273(2 Pt 1):L485-94. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flow cytometry and microscopy analyses have demonstrated that 9-nitrocamptothecin (9NC) induces apoptosis in prostate carcinoma LNCaP, DU-145 and PC-3 cells grown in culture or as xenografts. 9NC induces apoptosis regardless of the ability of the cells to induce tumors following xenografting into nude mice. Detection of apoptosis by flow cytometry was preceded or accompanied by increased cell size, loss of nuclear structure and vacuolization, as the tumor regressed, but no visible chromatin fragmentation. This is the first demonstration that 9NC is curative for human prostate carcinoma xenografts in the nude mouse model in the absence of detectable drug-induced toxicity during and after tumor regression. These findings indicate that 9NC may develop into a chemotherapeutic drug for the effective treatment of prostate cancer patients. Further, there was no apparent correlation of the steady-state level of the apoptosis-regulating proteins, Bcl-2, Bcl-XL, Bax and Ich-1, with tumorigenicity of the prostate cells xenografted in nude mice, aggressiveness of tumors grown in nude mice, and induction of apoptosis by 9NC. However, the TIAR protein was present at markedly high levels in all prostate carcinoma cell lines and this may correlate with their susceptibility to 9NC-induced apoptosis.
Journal of Experimental Therapeutics and Oncology 10/1996; 1(5):322-33.
[Show abstract][Hide abstract] ABSTRACT: Human leukemia U-937/WT cells were exposed to stepwise increased concentrations of Vincristine so that Vincristine-resistant cell sublines (termed U-937/RV) were developed. Established U-937/RV cell sublines have continuously propagated over a year, both in absence and presence of VCR, and have demonstrated similar features. In contrast to U-937/WT cells, U-937/RV cells have longer doubling time, and are more differentiated as determined by appearance of distinct morphological features and synthesis of mRNA that codes for the monocyte colony-stimulating factor-1 receptor (c-fms). Both apoptosis-suppressing Bcl-2 and Bcl-XL proteins were undectable in U-937/WT cells, whereas Bcl-2 was nearly detectable and Bcl-XL readily detectable in U-937/RV cells. The apoptosis-promoting Bax protein was also absent in U-937/WT cells and readily detected in U-937/RV cells. Vincristine-resistant cells with different levels of resistance synthesize similar levels of c-fms mRNA and Bax protein. Finally, unlike U-937/WT cells, U-937/RV cells have no ability to induce tumors when xenografted in immunodeficient mice. The findings collectively suggest that development of resistance to Vincristine in U-937/WT cells may correlate with cell differentiation and synthesis of proteins that regulate apoptosis.
European Journal Of Haematology 08/1996; 57(1):79-86. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell sublines resistant to doxorubicin (DOX) were developed from the human leukemia cell line, U-937/WT, exposed to stepwise DOX increases. In contrast to U-937/WT cells, the DOX-resistant U-937/RD cells have longer doubling time; are more differentiated along the monocytic lineage as determined by the presence of morphological features and mRNA coding for the monocyte colony-stimulating factor-1 receptor; synthesize the apoptosis-associated Bax protein; are less sensitive to apoptosis-inducing topoisomerase II-directed drugs, apparently because of increased synthesis of P-glycoprotein; and are practically non-tumorigenic when xenografted in nude mice. However, U-937/WT and U-937/RD cells exhibit similar sensitivity to the apoptosis-inducing drug 9-nitrocamptothecin. These findings suggest that several mechanisms are involved in the development of DOX-resistance in U-937 cells, and further, 9-nitrocamptothecin can overcome resistance to DOX. These findings may have clinical implications.
Anticancer research 01/1995; 15(5B):1873-81. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have investigated the possibility that some serum factors might negatively regulate the expression of the insulin-like growth factor-II (IGF-II) gene in 18-54, SF cells. Northern blot analyses indicated that there were three major transcripts (3.8 kb, 1.8 kb, and 1.2 kb) of the IGF-II gene in these cells. We found that incubation of 18-54, SF cells in medium containing very high concentrations (50-100%) of chicken serum greatly inhibited the steady-state level of all three IGF-II mRNA species. In addition, we also found that incubation of 18-54, SF cells in medium containing lower concentrations (10-50%) of chicken serum induced a 3.5 kb IGF-II mRNA. The inhibitory effect of high concentrations of chicken serum on IGF-II mRNA expression was not due to a cytotoxic effect of the serum, because these cells were maintained in 100% chicken serum for up to two weeks without loss of cell viability. The inhibitory effect of chicken serum on IGF-II mRNA was reversible after withdrawal of the serum. Nuclear run-on assays suggested that this negative regulation of IGF-II mRNA in 18-54, SF cells by chicken serum was not the result of transcriptional inhibition. Treatment of 18-54, SF cells that had been previously incubated in 100% chicken serum for 24 h with actinomycin D resulted in a partial restoration of the expression of the 3.8 kb and 1.2 kb IGF-II mRNA in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular and Cellular Endocrinology 04/1994; 99(2):293-300. · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study we evaluated the regulatory effect of 8-Cl-cyclic AMP (cAMP) on the proliferation of nontransformed cells. Our data indicate that similar to the reported inhibitory effect of 8-Cl-cAMP on the proliferation of Molt-4 lymphoma cells, the inhibitory effect of 8-Cl-cAMP on the proliferation of nontransformed fibroblast, myoblast and endothelial cells was dependent on serum or cyclic nucleotide phosphodiesterases and other nucleotide phosphatases. This suggested that the metabolism of 8-Cl-cAMP was required for the inhibition of cell proliferation. In addition, this serum-dependent inhibitory effect of 8-Cl-cAMP was antagonized by the presence of exogenous adenosine deaminase, suggesting that 8-Cl-adenosine was responsible for mediating the serum-dependent inhibitory effect of 8-Cl-cAMP on these cells. This idea was supported further by the ability of 8-Cl-adenosine to duplicate the inhibitory effect of 8-Cl-cAMP on cell proliferation in defined medium.
Journal of Pharmacology and Experimental Therapeutics 06/1993; 265(2):790-4. · 3.89 Impact Factor