The combination regimen of idarubicin and taxotere is effective against human drug-resistant leukemic cell lines.

Up-regulation of Bcl-2 protein may contribute to drug resistance, by decreasing apoptosis after treatment, in pre-B and B-cell leukemias in pediatric patients. By contrast, augmented caspase-3 activity, an effector caspase, may be indicative of drug sensitivity due to increased cellular apoptosis. We have reported the development of an in vitro human T-lymphoblastic leukemia model resistant to ara-C and/or native E. coli L-asparaginase (ASNase), mimicking the drug resistance to the Capizzi II regimen. We have investigated the potential drug synergism between Idarubicin (IDA) and Taxotere (TXR) that may be active in the ara-C and ASNase double drug-resistant cell lines. The additive or synergistic activity between IDA and TXR is drug concentration-dependent in inducing caspase-3 activation and cellular apoptosis. We exposed two human drug-resistant cell lines that do not express the MDRI phenotype, one resistant to ASNase alone (CEM/ASNase-1-3) and the other resistant to both ara-C and ASNase (CEM/ara-C/I/ASNase-0.5-2), to physiologically achievable concentrations of IDA, TXR, or their combination. Both lines showed either synergistic drug activity to the combination regimen in comparison to either drug used alone, as determined by MTT assay, or additivity as demonstrated by flow cytometry after Annexin V and propidium iodide (PI) staining. After exposure of the ASNase-resistant line to various concentrations, the intracellular levels of Bcl-2 protein decreased to near zero relative to untreated control cells. The Bcl-2 protein reductions in these cells ranged from 30% to <1%, 49% to <1%, and 27% to 3% when treated with IDA or TXR as a single drug or IDA + TXR combination, respectively. Similarly, intracellular Bcl-2 levels in the double-resistant cell line decreased with reductions ranging from 24% to <1%, 87% to <1%, and 46% to <1% of the untreated control after treatment with IDA, TXR, or their combination, respectively. Conversely, the caspase-3 activity increased in a dose-dependent manner and inversely-correlated with loss of cell viability (r= 0.91) after exposure to IDA + TXR combination in the double drug-resistant line to both ara-C and ASNase. We conclude that the combination of the IDA + TXR regimen is highly synergistic or additive in drug resistant human leukemic cell clones. The molecular mechanism of action is due to the down-regulation of Bcl-2 protein and up-regulation of caspase-3 activity. This drug combination warrants further investigation for use in the treatment of patients with ara-C and/or ASNase refractory leukemias.

Taxotere and vincristine inhibit the secretion of the angiogenesis inducing vascular endothelial growth factor (VEGF) by wild-type and drug-resistant human leukemia T-cell lines.

Recent studies have shown that angiogenesis, which is induced by VEGF, may be involved in the pathogenesis of hematopoietic malignancies. A human leukemia model consisting of T-lymphoblastic CEM/0, 7 monoclonal refractory clones resistant to both cytosine arabinoside (ara-C) and L-asparaginase (ASNase), Jurkat/E6-1 and U937, representing the leukemic blasts from relapsed patients with leukemias was investigated for secretion of VEGF before and after treatment with various agents. The T-lymphoblastic cell line, Jurkat/E6-1, was used as the negative control, which has been characterized as not expressing mRNA nor the VEGF protein, and did not secrete VEGF. With no treatment, U937, the positive control, secreted the highest VEGF concentration of 1612.7 pg/ml. The CEM/O wild type cell line and 5 other drug-resistant clones secreted VEGF at levels ranging from 180.9 to 414.2 pg/ml. Two CEM drug-resistant clones, CEM/ara-C/G/ASNase-0.5-1 and CEM/ara-C/G/ASNase-1-1, lacked VEGF production. Docetaxel (Taxotere, TXR), Vincristine (VCR), ASNase, and the Fit-1/Fc chimera, a specific inhibitor of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, were tested for inhibition of VEGF secretion. Treatment of the leukemic cell lines with 2 microg/ml Flt-1/Fc chimera for 24 hours completely inhibited VEGF secretion to the detection limit of the assay (<10pg/ml). After 24 hours incubation with Flt-1/Fc chimera, the leukemic cells appeared to be undergoing apoptosis, based on microphotography examination, suggesting that VEGF could be used in an autocrine loop to promote cell survival by the leukemic cells. Treatment with 0.5, 1, and 2 microg/ml Flt-1/FC chimera for 48 hours demonstrated a 15-25% growth inhibition by MTT assay. Strong inhibition of VEGF secretion in the culture media was observed after 10 microM TXR or 0.1 microM VCR for 24 hours in the wild-type and drug-resistant clones, except CEM/ara-C/I, in comparison with controls. In contrast, treatment with 1 IU/ml ASNase, a specific T-cell protein inhibitor, in 5 cell lines for 24 hours demonstrated no inhibition of VEGF in CEM/0 3 drug-resistant clones and the myeloid U937 line. We conclude that the leukemia cell lines actively secrete VEGF, in vitro. TXR and VCR, but not ASNase, strongly inhibit the VEGF production, suggesting that inhibition of this growth factor may be a mechanism of antileukemic activity. Moreover, the leukemic cell lines examined here may constitute a useful model to study antiangiogenic drugs, alone or in combination with established drug regimens used against refractory leukemias.

Synergistic antiviral effect of PEG-asparaginase (ONCASPAR), with protease inhibitor alone and in combination with RT inhibitors against HIV-1 infected T-cells: a model of HIV-1-induced T-cell lymphoma.

We evaluated the anti-HIV-1 activity of the T-cell-specific protein inhibitor PEG-asparaginase (PEG-ASNase) in human HIV-1-infected T-cells. We further examined the drug synergism between PEG-ASNase and the protease inhibitor Saquinavir (SAQ), both alone and in combination with nucleoside analog reverse transcriptase inhibitors (NRTI). Our drug synergism studies served as a model for an HIV-induced T-cell lymphoma. Phytohemagglutinin [PHA(+)] stimulated T-cells were infected with HIV-1 and then treated with one or more drugs 90 minutes from the viral exposure. To measure inhibition of viral replication, we examined HIV-1 RT and HIV-1 RNA in the supernatant and intracellularly on day 7 post-infection and drug treatment. Last, we examined the effect of administering drugs immediately after HIV-1 infection of T-cells to simulate treatment after an accidental exposure to the virus. PEG-ASNase, even when used alone, has anti-HIV-1 activity in PHA(+)-stimulated T-cells due to inhibition of protein synthesis. When the drug was used with SAQ, the combination was synergistic in inhibiting HIV-1 RT and RNA in the supernatant and intracellularly by 2.5 log10 in comparison with controls. PEG-ASNase and SAQ were even more effective in inhibiting HIV-1 replication when combined with the NRTI inhibitors azidothymidine (AZT) and (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine). The addition of ribonucleotide reductase inhibitor, 2-methyl-1H-isoindole-1,3-dione (MISID), further potentiated the antiviral effect of the regimen. HIV-1 RT and RNA analyses showed that the administration of the PEG-ASNase + SAQ drug combination immediately following exposure to HIV-1 completely inhibited the infection of T-cells in our in vitro T-cell model. From these results we conclude that PEG-ASNase is a valuable T-cell-specific protein inhibitor against HIV-1 infection, when used singly or in combination with a protease inhibitor, an RT inhibitor and an RR inhibitor. Since PEG-ASNase is a drug of choice for the treatment of T-cell lymphomas, a combination regimen containing PEG-ASNase could be very effective in the treatment of HIV-1-induced T-cell lymphoma and possibly AIDS. Future studies are needed in HIV-infected and/or HIV-induced T-cell lymphoma patients to investigate these findings.

Development of a double-drug-resistant human leukemia model to cytosine arabinoside and L-asparaginase: evaluation of cross-resistance to other treatment modalities.

We have developed an in vitro model of 38 T-lymphoblastic leukemia lines resistant to cytosine arabinoside (ara-C) and L-asparaginase (ASNase). Of these, 26 cell lines resistant to both drugs, 6 resistant to ara-C, and 6 resistant to ASNase were isolated. In 18 of these cell lines, all randomly selected, resistance to ara-C, ASNase and gamma radiation was documented by the MTT and trypan blue assays, as well as flow cytometry with Annexin V and propidium iodide (PI) staining. In these lines, p53, p21WAF1, and bcl-2 levels were measured by ELISA. Results show that P21WAF1 upregulation following p53 induction did not occur, suggesting that p53 function may be lost. Moreover, the data imply that upregulation of bcl-2 is critical in the development of resistance to ara-C and ASNase in these leukemic lines. In the CEM/0 parent line, p53 maintained its ability to interact with its DNA binding site as documented by the electrophoretic mobility shift assay (EMSA). But in one single- and one double-resistant leukemic cell line examined, p53 was not shown to maintain this ability. We conclude that double-resistant clones to ara-C and ASNase are refractory to both drugs, providing an excellent leukemic model to investigate the multiple-drug resistance.

Increased p21/WAF-1 and p53 protein levels following sequential three drug combination regimen of fludarabine, cytarabine and docetaxel induces apoptosis in human leukemia cells.

Combinations of nucleoside analog drugs, such as F-araA and ara-C, combined with Topoisomerase II inhibitors, such as anthracyclines, are synergistic against human leukemic T-cells and induce apoptotic cell death. Similarly, nucleoside analog drugs followed by mitotic inhibitors also have a synergistic effect. Sequence specific combinations of F-araA followed by ara-C and Taxotere (docetaxel) in CEM/0 cells showed a 2- to 3-fold synergism over the two drug (F-araA + ara-C) combinations and 2- to 4-fold synergism over Taxotere alone. This synergism was evident due to enhanced cellular apoptosis. In the CEM/ara-C/7A cell line, which is partially resistant to ara-C, the synergy observed with the triple drug combination was 9-fold greater than the F-araA plus araC combination, and 3-fold greater than Taxotere alone, making this three-drug regimen collaterally sensitive to ara-C. This study describes the mechanisms of the synergistic effect in regards to apoptosis achieved by three-drug regimens comprised of two nucleoside analog drugs and a mitotic inhibitor in comparison with the combination of two nucleotide analog drugs. The study also demonstrates that the possible biochemical mechanism of cellular toxicity and drug synergism is attributed to induction of apoptosis following drug treatment and the onset of the apoptotic cascade is primarily regulated by p21/WAF-I, which is transcriptionally activated by p53 following DNA damage. The anti-apoptotic protein, bcl-2, seemed to have no effect in inhibiting apoptosis following treatment with the two or three drug regimens in this in vitro leukemia model. The three-drug combination induced greater cellular apoptosis than the two-drug combination or Taxotere monotherapy. We conclude that the greater drug synergism observed in human leukemic cells, sensitive or resistant to ara-C, by Fludarabine + ara-C + Taxotere can be explained by the greater oligonucleosomal DNA fragmentation indicative of increased cellular apoptosis. The mechanism of this increased cytotoxic action is due to the upregulation of p53 and p21/WAF-1 with a down regulation of bcl-2. These studies are encouraging, and testing this three drug regimen in a clinical setting may result in improved antileukemic therapies.

Development of zidovudine (AZT) resistance in Jurkat T cells is associated with decreased expression of the thymidine kinase (TK) gene and hypermethylation of the 5' end of human TK gene.

The T-cell line Jurkat E6-1 was rendered resistant to zidovudine (AZT) in vitro by exposure to low but gradually increased concentrations of the drug. Biochemical pharmacology studies of [3H]AZT in the AZT-resistant T-cell lines showed a significant reduction of AZT phosphorylation to the mono-, di-, and triphosphate anabolites. Peripheral blood mononuclear cells (PBMCs) from pediatric patients with human immunodeficiency virus type 1 (HIV-1) infection showed a similar pattern of decreased AZT anabolism. Enzymatic studies with purified thymidine kinase (TK) preparations from these cell lines showed a gradual decline in Vmax related to their level of resistance to AZT. The Jurkat/AZT-20 and Jurkat/AZT-100 cells were studied in greater detail with reverse transcriptase/polymerase chain reaction (RT/PCR) cloned probes to determine possible molecular mechanisms of resistance to AZT. TK mRNA was significantly decreased (approximately 5- to 10-fold) in the AZT-resistant T-cell lines. Southern blot analyses indicated that there were no major rearrangements or deletions of the TK gene, but the 5' end of the gene in the AZT-resistant cells is highly methylated when compared to wild-type cells. No apparent differences were seen in thymidylate kinase (dTMPk) mRNA levels in the same T-cell lines. Thus the decreased expression of TK mRNA and resultant TK enzymatic activity is responsible for the observed reduction in the AZT anabolism in the resistant T-cell lines. Decreased T-cell TK activity could allow wild-type, AZT-sensitive HIV-1 to replicate in the presence of subinhibitory AZT triphosphate (AZT-TP) cellular concentrations enabling a genetic variant with drug resistance to emerge and outgrow the AZT-sensitive, wild-type virus.

Evidence of in vitro development of drug resistance to azidothymidine in T-lymphocytic leukemia cell lines (Jurkat E6-1/AZT-100) and in pediatric patients with HIV-1 infection.

Clinical reports indicate that the development of drug resistance to AZT after chronic administration is common. In order to study this phenomenon, the T-cell line Jurkat E6-1 was treated continuously in vitro with low, gradually increased, concentrations of azidothymidine (AZT). Initially, 1 microM AZT significantly retarded the cell line from reaching confluence. However, after 10 weeks the T-cell line was able to grow in 10 microM AZT without any evidence of growth inhibition. Subsequently, cell isolates could grow continuously in the presence of 20, 50, and 100 microM AZT without growth inhibition. These T-cell lines (Jurkat E6-1/AZT-10, Jurkat E6-1/AZT-20, Jurkat E6-1/AZT-50, and Jurkat E6-1/AZT-100) were tested for AZT anabolism using purified [3H]AZT, and the results were compared to the wild-type untreated Jurkat E6-1 cell line. Similar intracellular AZT anabolites concentrations were determined in all cell lines. However, a four- to sixfold lower cellular concentration of mono-, di-, and triphosphate anabolites of AZT was determined in the Jurkat E6-1/AZT-10 cell line after 1 microM AZT incubation and 6.5-fold lower after 10 microM AZT treatment. In general, a five- to sixfold reduction in the phosphorylation rates were estimated in the AZT resistant T-cell line. Pharmacology studies of [3H]AZT in the Jurkat E6-1/AZT-100 cell line showed a much lower level of activation of the pro-drug (28-fold), due to lack of thymidine kinase (TK) activity when compared to the Jurkat E6-1/AZT-10 T-cell line. A similar level of resistance was obtained at the thymidylate (dTMP) kinase level. Concurrently an additional mode of resistance (407-fold) was seen on the incorporation of the AZT triphosphate anabolite (AZTTP) into cellular DNA. The formation of this cell line in a period of < or = 4 months coincides with the evidence of the clinical development of "resistance" to AZT in patients who receive the drug continuously. In addition, these T-cell lines have been infected with HIV, and studies on the development of collaterally sensitive regimens are under way.

Retention and distribution of iron added to cow's milk and human milk as various salts and chelates.

Iron supplementation of infant formulas is recommended by most national and international organizations, but the optimal form of supplementation has not been determined. We have compared the bioavailability and tissue distribution of iron from four iron chelates and two commonly used iron salts. Weanling C57BL/6J mice were fed for 1 week an evaporated cow's milk diet supplemented with vitamins and minerals (except for iron). Following the adjustment period, mice were divided into 12 groups of 20 each. Six groups continued to receive the cow's milk diet for 18 hours, while the other six groups were fed a similar diet based on human milk. Individual groups received a single dose of milk radioactively labeled with Fe(II)Cl2, Fe(II)SO4, Fe(III)NTA, Fe(III)EDTA, Fe(III)citrate or Fe(III)lactobionate. Wholebody retention was measured after 4 days; animals were then killed and individual tissues were counted for radioactivity. Iron from FeCl2, FeSO4 and FeNTA were the best retained from both milk diets. Fe citrate had a significantly lower iron retention than all other groups in either diet and is probably not an effective chelate for delivering iron to milk diets. Iron bioavailability was higher from the human milk diets than from the cow's milk diets from all vehicles used except citrate and lactobionate. Absorption of Fe citrate was similar from the two milk diets, while percent retention from Fe lactobionate was higher from cow's milk than from human milk. Tissue distribution of retained iron was similar for the milk diets and among the groups, indicating that, once absorbed, iron from the different vehicles is metabolized in a similar manner.