Biomedical subjects

P Pantazis

Publications and source records attributed to P Pantazis.

At least 37 records · Page 2Linked to original sources

PubMed1999

Propionate and butyrate esters of camptothecin and 9-nitrocamptothecin as antileukemia prodrugs in vitro.

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.

Animals↗

PubMed1999

Cif (Cytochrome c efflux-inducing factor) activity is regulated by Bcl-2 and caspases and correlates with the activation of Bid.

The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochrome c from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals.

Apoptosis↗

PubMed1998

9-Nitrocamptothecin inhibits tumor recrosis factor-mediated activation of human immunodeficiency virus type 1 and enhances apoptosis in a latently infected T cell clone.

Transition from latency to active replication is a crucial stage for the process of human immunodeficiency virus type 1 (HIV-1) infection and life cycle. HIV-1 replication in latently infected cells can be strongly induced by the cytokine tumor necrosis factor alpha (TNF-alpha) and the proliferation-arresting chemical sodium butyrate (NaB). We have investigated the ability of the drug 9-nitrocamptothecin (9NC), a potent cellular topoisomerase I (topo I) inhibitor currently in clinical trials in cancer patients, to regulate HIV-1 replication in latently infected lymphocytic ACH-2 cells on reactivation with either TNF-alpha or NaB. Treatment of ACH-2 cells with 9NC alone resulted in increased levels of viral transcripts, while there was a slight reduction or no change in the levels of host cell transcripts. However, pretreatment of ACH-2 cells with 9NC inhibited TNF-alpha-induced extracellular HIV-1 p24 levels up to approximately 95% and nearly 80% of the cell-associated viral RNAs. The quantitative decrease in viral products was concomitant with a decrease in cellular gene expression and induction of apoptosis in the host cells. 9NC blocked the infected cells at the boundary of the S and G2 phases, resulting in an accelerated apoptosis that was further enhanced with TNF-alpha treatment. Similar results were observed following concurrent exposure to TNF-alpha and 9NC, but 9NC failed to inhibit upregulation of HIV-1 mRNA in ACH-2 cells exposed to TNF-alpha before 9NC treatment. Further, 9NC had no inhibitory effect on NaB-induced apoptosis and upregulation of HIV-1 mRNA expression regardless of whether 9NC and NaB were used concurrently or in various treatment sequences. In uninfected lymphocytic CEM cells derived from a common parental cell line, a slight downregulation of cellular gene expression was detected along with low-level apoptosis. These results demonstrate that 9NC impairs TNF-alpha-induced, but not NaB-induced, HIV-1 activation, and suggest a means of inhibiting active HIV-1 viremia arising as a result of elevated TNF-alpha levels.

Antineoplastic Agents↗

PubMed1998

Altered sensitivities to anticancer and differentiation agents in etoposide-resistant human myeloid leukemia U-937 cells.

We previously have exposed U-937 human leukemia cells to stepwise increased concentrations of the anticancer drug etoposide, and this treatment has resulted in stable sublines (termed U-937/RE) exhibiting various extents of resistance to the drug and constitutively expressing c-fms mRNA, a specific marker of monocytic differentiation. In this report, we pursued studies to show that the P-glycoprotein blocker, verapamil, partially restores sensitivity to etoposide in U-937/RE cells. Further, the U-937/RE cells exhibit differential sensitivities to compounds that induce maturation of U-937 cells, as judged by the ability to reduce nitroblue tetrazolium and by morphologic changes, and increased sensitivities to apoptosis induction by the cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) and the anticancer drugs 9-nitrocamptothecin and doxorubicin. In addition, the U-937/RE cells, xenografted in immunodeficient mice, demonstrate decreased or no ability to induce tumors. Taken together, these findings indicate that U-937/RE cells differ from the parental U-937 cells in several functional properties and can serve as models to develop protocols for treatment of human leukemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Acquisition of cellular resistance to 9-nitro-camptothecin correlates with suppression of transcription factor NF-kappa B activation and potentiation of cytotoxicity by tumor necrosis factor in human histiocytic lymphoma U-937 cells.

Resistance of tumor cells to chemotherapeutic agents is a major problem in cancer therapy. Continuous exposure of human histiocytic lymphoma U-937 cells to 9-nitro-camptothecin (9NC), an inhibitor of the nuclear DNA topoisomerase I, induces resistance to this drug. Because of the involvement of the nuclear factor NF-kappa B in the expression of several growth regulatory genes, we examined the activation of this transcription factor in 9NC-resistant U-937 cells. We found that resistance to increasing concentrations of 9NC correlated with resistance to tumor necrosis factor (TNF)-dependent activation of NF-kappa B. However, the constitutive synthesis of NF-kappa B proteins remained unaffected. Cellular resistance was not unique to TNF, as other activators of NF-kappa B, including interleukin-1, phorbol ester and hydrogen peroxide, also had no effect. There was no difference between 9NC-sensitive and -resistant cells in the activation of NF-kappa B by okadaic acid. Other transcription factors, including AP-1 and Oct-1, were not affected in the resistant cells. When examined for the inhibitory subunit of NF-kappa B (I kappa B alpha), resistant cells showed a faster rate of resynthesis than the control. Interestingly, although 9NC resistance correlated with resistance to TNF-dependent NF-kappa B activation, TNF-dependent cytotoxicity in these cells was enhanced by several hundred fold despite a significant decrease in the number of TNF receptors. In conclusion, our results suggest that NF-kappa B activation may play a role in tumor cell killing by 9NC but not by TNF.

Antineoplastic Agents↗

PubMed1997

Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning.

Camptothecins are a new class of anticancer drugs that target DNA topoisomerase I; current efforts are directed toward elucidating optimal combinations of these drugs with other antineoplastic agents. A rationale for the use of sequential therapy involving the combination of camptothecins with topoisomerase II-targeting drugs, such as etoposide, has arisen from observations of increased topoisomerase II protein levels in cell lines resistant to camptothecin. In an effort to understand potential mechanisms of resistance to this strategy, we developed a U-937 cell subline, denoted RERC, that is capable of surviving exposure to sequential topoisomerase poisoning. The RERC cells are 200-fold resistant to camptothecin, 8-fold resistant to etoposide, and 10-fold hypersensitive to cisplatin compared to the parental U-937 cells. Biochemical analyses indicate that the resistant phenotype involves alterations in both topoisomerase I and topoisomerase IIalpha. Topoisomerase I catalytic activity in the resistant cells is similar to that of the parental line but is resistant to camptothecin. Moreover, the resistant cells express a single mRNA species of topoisomerase I that codes for a mutation in codon 533. In addition, topoisomerase IIalpha protein levels are decreased 10-fold in the resistant line, coincident with a two-fold decrease in the expression of topoisomerase IIalpha mRNA. Collectively, these results indicate that resistance to sequential topoisomerase poisoning may involve a reduction in total cellular topoisomerase activity.

Antineoplastic Agents↗

PubMed1997

Integration of proviral DNA into the PDGF beta-receptor gene in HTLV-I-infected T-cells results in a novel tyrosine kinase product with transforming activity.

We have previously shown that noninfected human T-cell lines express the canonical 5.7 kb mRNA coding for the type beta platelet-derived growth factor-receptor (PDGF beta-receptor), whereas HTLV-I-infected T-cell lines express a novel PDGF beta-receptor mRNA of 3.8 kb. In this report, we have extended those studies to molecularly characterize the 3.8 kb PDGF beta-receptor mRNA and show that it has resulted from integration of an apparently undeleted HTLV-I provirus into the PDGF beta-receptor gene in an orientation enabling expression of a truncated PDGF beta-receptor mRNA using the 3' HTLV-I long terminal repeat as a promoter. Further, NIH3T3 cells transfected with a plasmid containing the truncated PDGF beta-receptor ORF plasmid generate colonies in soft agar with more cells per colony than untransfected cells, or cells transfected with the Tax 1 or PDGF-B (c-sis) plasmids. These results indicate that the truncated PDGF beta-receptor protein acquires transforming capability and that HTLV-I-induced truncation of PDGF beta-receptor may correlate with HTLV-I-associated neoplasia of human T-cells.

3T3 Cells↗

PubMed1997

Antiproliferative effect of curcumin (diferuloylmethane) against human breast tumor cell lines.

Pharmacologically safe compounds that can inhibit the proliferation of tumor cells have potential as anticancer agents. Curcumin, a diferuloylmethane, is a major active component of the food flavor turmeric (Curcuma longa) that exhibits anticarcinogenic properties in vivo. In vitro, it suppressed c-jun/Ap-1 and NF-kappaB activation and type 1 human immunodeficiency virus long-terminal repeat-directed gene expression. We examined the antiproliferative effects of curcumin against several breast tumor cell lines, including hormone-dependent and -independent and multidrug-resistant (MDR) lines. Cell growth inhibition was monitored by [3H]thymidine incorporation, Trypan blue exclusion, crystal violet dye uptake and flow cytometry. All the cell lines tested, including the MDR-positive ones, were highly sensitive to curcumin. The growth inhibitory effect of curcumin was time- and dose-dependent, and correlated with its inhibition of ornithine decarboxylase activity. Curcumin preferentially arrested cells in the G2/S phase of the cell cycle. Curcumin-induced cell death was neither due to apoptosis nor to any significant change in the expression of apoptosis-related genes, including Bcl-2, p53, cyclin B and transglutaminase. Overall our results suggest that curcumin is a potent antiproliferative agent for breast tumor cells and may have potential as an anticancer agent.

Antibiotics, Antineoplastic↗

PubMed1997

Monocytic differentiation of HL-60 promyelocytic leukemia cells correlates with the induction of Bcl-xL.

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.

Apoptosis↗

PubMed1996

Induction of apoptosis in malignant and camptothecin-resistant human cells.

Flow cytometry studies demonstrate that androgen-independent human prostate carcinoma DU-145 cells are arrested at the G1-phase of the cell cycle in the presence of suramin, but they die by apoptosis in the presence of 9-nitrocamptothecin (9NC). The addition of cytostatic concentrations of suramin increases the apoptotic action of 9NC on DU-145 cells, and induces apoptosis in 9NC-resistant DU-145/RC cells that were derived from the parental DU-145 cells by continuous exposure to progressively increased concentrations of 9NC. In addition, the topoisomerase II-directed drug etoposide exerts more extensive apoptotic action on DU-145/RC than DU-145 cells. Increased resistance of DU-145 cells to 9 NC and collaterally increased sensitivity to etoposide and suramin appear to correlate with alterations in the structure rather than synthesis of topoisomerases and possibly with specific cellular proteins that regulate apoptosis. The results suggest that etoposide and suramin may be successful alternative treatments for 9NC-resistant androgen-independent prostate cancer.

Androgens↗

PubMed1996

Combination immunotoxin treatment and chemotherapy in SCID mice with advanced, disseminated Daudi lymphoma.

We describe the use of an immunotoxin (IT) cocktail (anti-CD22- and anti-CD19-ricin A chain) and any 1 of 3 chemotherapeutic drugs (doxorubicin, cytoxan or camptothecin) to treat advanced disseminated Daudi lymphoma in SCID mice (SCID/Daudi). In a previous report, we demonstrated that this regimen was curative when given the day following tumor cell inoculation. Here, we show that combination therapy in mice with advanced tumor significantly increased their survival, although it was not curative. Importantly, the outcome of therapy was dependent upon the temporal order in which IT and chemotherapy were administered. Thus, the best anti-tumor effect was achieved when an IT cocktail was given before or at the same time as chemotherapy. When the IT was given after chemotherapy, there was no additional therapeutic benefit. Our results confirm the rationale of using combination therapy in the treatment of advanced B-cell neoplasia and suggest that ITs should be administered prior to or during chemotherapy.

Animals↗

PubMed1996

Isolation and characterization of an apoptosis-resistant variant of human leukemia HL-60 cells that has switched expression from Bcl-2 to Bcl-xL.

Human promyelocytic leukemia HL-60 cells treated with 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) undergo growth arrest and subsequently die by apoptosis. We describe here the isolation of a variant of HL-60 cells, HCW-2, which was resistant to the cytotoxic effects of 8-Cl-cAMP, but still underwent growth arrest. Thus, HCW-2 cells appeared to be altered in their ability to undergo apoptosis. HCW-2 cells were also completely refractory to the apoptotic action of cycloheximide and staurosporine, two compounds which were very potent inducers of apoptosis in the parental HL-60 cells, suggesting that the resistance to apoptosis was not unique to 8-Cl-cAMP. Western blot analysis demonstrated that the parental HL-60 cells expressed both Bcl-2 and Bax, two factors known to be intimately involved in the control of apoptosis. Surprisingly, HCW-2 cells no longer expressed Bcl-2 protein and paradoxically contained Bax protein at a level that was approximately 50-fold higher than in HL-60 cells. However, Northern and Western analyses indicated that the apoptotic suppressor gene, bcl-xL, which is not expressed in the parental HL-60 cells, was expressed in HCW-2 cells. Thus, the Bcl-2-independent resistance of HCW-2 cells to apoptotic induction is discussed in terms of the expression of bcl-xL.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Monocytic differentiation and synthesis of proteins associated with apoptosis in human leukemia U-937 cells acquiring resistance to vincristine.

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.

Animals↗

PubMed1996

Establishment of human prostate tumor xenografts in nude mice and response to 9-nitrocamptothecin in vivo and in vitro does not correlate with the expression of various apoptosis-regulating proteins.

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.

Animals↗

PubMed1995

Sensitivity of camptothecin-resistant human leukemia cells and tumors to anticancer drugs with diverse mechanisms of action.

Human leukemia U-937 cell clones resistant to 9-nitrocamptothecin (9NC) appear after exposure to increase 9NC-concentrations. Drug resistance is irreversible, regardless of whether the 9NC-resistant (U-937/CR150) cells grow in media with or without 9NC. U-937/CR150 cells are more sensitive than wild type U-937 (U-937/wt) cells to topoisomerase II-directed drugs, amsacrine, daunorubicin, and etoposide. The mitotic inhibitor, vincristine, induces hyperdiploidy in U-937/wt, but not in U-937/CR150 cells, whereas the antimetabolites, cytarabine and methotrexate, and the nitrosourea, carmustine, elicit similar responses in both U-937/wt and U-937/CR150 cells. U-937/CR150-generated tumors in nude mice are sensitive to etoposide. The clinical implications of increased sensitivity of 9NC-resistant tumors to some anticancer drugs are discussed.

Animals↗

PubMed1995

The water-insoluble camptothecin analogues: promising drugs for the effective treatment of haematological malignancies.

After failing to exhibit benefits in clinical studies with cancer patients in the early 1970s, camptothecin (CPT) and its water-insoluble analogues are re-emerging as promising drugs with multiple actions in the treatment of human haematological malignancies. CPT analogues interfere with the mechanism of action of the nuclear enzyme topoisomerase I, while the cells progress through the S-phase of the cell cycle and this results in cell death by apoptosis. Modulations of topoisomerase I phosphorylation may indirectly modulate the cytotoxic activity of CPT analogues. In vitro, CPT analogues have exhibited increased or unaltered killing activity against leukaemia cells resistant to epipodophyllotoxins, anthracyclines, anthracenediones, and Vinca alkaloids, while development of resistance to CPT analogues renders leukaemia and lymphoma cells more sensitive to topoisomerase II-directed drugs, inducers of cell differentiation, and immunotoxins. Oral administration of the CPT analogues has circumvented the inconvenience of solubility of these drugs. Metabolic conversion of the CPT analogue 9-nitro-CPT to equally or more potent 9-amino-CPT practically makes unnecessary treatment of the patient with 9-amino-CPT, which, in addition, is costlier to prepare than 9-nitro-CPT. Considering the therapeutic, economic and handling viewpoints, the overall conclusion is that the water-insoluble CPT analogues are very promising antileukaemia/antilymphoma agents that warrant further preclinical and clinical studies.

Animals↗

PubMed1995

The role of pH and serum albumin in the metabolic conversion of 9-nitrocamptothecin to 9-aminocamptothecin by human hematopoietic and other cells.

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Antineoplastic Agents↗

PubMed1995

Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation.

The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradiation. We demonstrate that the decision to undergo apoptosis in this cell line appears to be made at a G2 checkpoint. In addition, we characterize an HL-60 variant, HCW-2, which is radioresistant. HCW-2 cells display DNA damage induction and repair capabilities identical to those of the parental HL-60 cell line. Thus, the difference between the two cell lines appears to be that X-irradiation induces apoptosis in HL-60, but not in HCW-2, cells. Paradoxically, HCW-2 cells display high levels of expression of bax, which enhances apoptosis, and no longer express bcl-2, which blocks apoptosis. HCW-2 cells' resistance to apoptosis may be due to the acquisition of expression of bcl-XL, a bcl-2-related inhibitor of apoptosis. In summary, apoptosis can be induced in X-irradiated HL-60 cells by a p53-independent mechanism at a G2 checkpoint, despite the presence of endogenous bcl-2. The resistance shown by HCW-2 cells suggests that bcl-XL can block this process.

Apoptosis↗