Panayotis Pantazis

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.

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.

Conversion of 9-nitro-camptothecin to 9-amino-camptothecin by human blood cells in vitro.

To the Editor: In recent reports, we have shown that the compound 9-nitro-camptothecin (9NC) is cytostatic or cytotoxic for human cells in vitro, depending on the ability of these cells to induce tumors when xenografted into nude mice (1-5). Specifically, cells that are not tumorigenic in nude mice accumulate at late-S/G2 phase of the cell cycle, in presence of 9NC, and remain arrested for a prolonged period of time or re-enter cell cycling upon cessation of exposure to 9NC. In contrast, tumorigenic cells die by apoptosis while they traverse the S phase in the presence of 9NC, and cell death continues regardless of discontinuation of exposure to the drug. Also, we have shown that 9NC suppresses growth of and induces regression of human tumors established in nude mice

9-nitro-camptothecin delays growth of U-937 leukemia tumors in nude mice and is cytotoxic or cytostatic for human myelomonocytic leukemia lines in vitro

Abstract:  The camptothecin derivatives 9‐nitro‐camptothecin (9NC) and 9‐amino‐camptothecin (9AC) inhibit similarly growth of HL‐60, KG‐1, and U‐937 cells in vitro, whereas growth of THP‐1 cells is inhibited by 9AC, but not by 9NC. Growth inhibition is accompanied by enlargement of cells which contain one (HL‐60, THP‐1) or more (KG‐1, U‐937) nuclei. Flow cytometry studies showed that 9NC‐treated HL‐60 and U‐937 cells accumulate in the S and G2 phases of the cell cycle; then they die by apoptosis, with the HL‐60 cells being more sensitive than U‐937 cells to 9NC. In contrast, 9NC‐treated KG‐1 and THP‐1 cells accumulate in S and G2 phases, but resist death by apoptosis. Of the cell lines tested, only U‐937 cells xenografted in nude mice generated subcutaneous myeloid tumors, which exhibited a delayed growth in the presence of 9NC. Further, 9NC‐treated advanced U‐937 tumors regressed temporarily, indicating that U‐937 cells consist of 9NC‐sensitive and 9NC‐resistant populations.

Induction of Apoptosis in Malignant and Camptothecin‐resistant Human Cellsa

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.

Partial characterization of human leukemia U-937 cell sublines resistant to 9-nitrocamptothecin P

Abstract: Human leukemia U‐937 cell sublines exhibiting various levels of resistance to 9‐nitrocamptothecin (9NC) were developed after exposure to progressively increased 9NC concentrations. Increases in 9NC resistance of the cells were accompanied by decreases in proliferation rate; appearance of morphological and functional features that correlate with granulocytic maturation; decreased synthesis of topoisomerase I; increased synthesis of topoisomerase II; and inability or decreased ability to induce tumors when xenografted in nude mice. 9NC‐resistant cells, transferred and propagated in 9NC‐free media for 6 months, continue to exhibit resistance and other features similar to cells propagated in continual presence of 9NC. Finally, 9NC‐resistant U‐937 cells respond to physiological and non‐physiological agents of cell differentiation, indicating that alternative treatments can be successfully used to inhibit growth of 9NC‐resistant U‐937 cells and tumors.

Camptothecin: A Promising Antiretroviral Drug.

The plant alkaloid camptothecin (CPT) has demonstrated the ability to inhibit replication of the equine anemia virus (E1AV) and the human immunodeficiency virus (HIV) in infected cells in culture. Further, CPT prevented the development of lymphoma and erythroleukemia in mice infected with the Moloney murine leukemia virus and the Friend erythroleukemia virus, respectively, as assessed by prevention or reduction of splenomegaly. These results were observed at concentrations that had no apparent toxic effects on the mice. It has been suggested that the antiretroviral activity of CPT is mediated by the host cells enzyme topoisomerase I. Taken collectively, the findings indicate that CPT analogues may develop into potent drugs against various human and animal diseases caused by diverse retroviruses. Copyright 1996 S. Karger AG, Basel

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.

Monocytic differentiation and synthesis of proteins associated with apoptosis in human leukemia U‐937 cells acquiring resistance to Vincristine

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 colonystimulating 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.