Triazene compounds in the treatment of acute myeloid leukemia: a short review and a case report.

Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG). If not repaired, O(6)-MeG frequently mispairs with thymine during DNA duplication. O(6)-MeG:T mismatches can be recognized by the mismatch repair (MMR) system which activates a cascade of molecular events leading to cell cycle arrest and cell death. If MMR is defective, cells continue to divide and GC → AT transition mutations occur. In preclinical models, such mutations can lead to the appearance of abnormal proteins containing non-self peptides ("chemical xenogenization" CX) that can be recognized by host cell-mediated immunity. Repair of O(6)-MeG is achieved by the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), which removes the methyl adduct in an autoinactivating stoichiometric reaction. High MGMT levels attenuate the pharmacodynamic effects of triazenes. In the last few years, triazenes, alone or with MGMT inhibitors, have been tested in AML. In view of their potential activity as CX inducers, triazenes could offer the additional advantage of host anti-leukemia immune responses. The present paper describes several studies of leukemia treatment with triazenes and a case of acute refractory leukemia with massive skin infiltration by malignant cells. Treatment with Temozolomide and Lomeguatrib, a potent MGMT inhibitor, produced a huge, although transient, blastolysis and complete disappearance of all skin lesions.

Interfacing Sca-1(pos) mesenchymal stem cells with biocompatible scaffolds with different chemical composition and geometry.

An immortalized murine mesenchymal stem cell line (mTERT-MSC) enriched for Lin(neg)/Sca-1(pos) fraction has been obtained through the transfection of MSC with murine TERT and single-cell isolation. Such cell line maintained the typical MSC self-renewal capacity and continuously expressed MSC phenotype. Moreover, mTERT-MSC retained the functional features of freshly isolated MSC in culture without evidence of senescence or spontaneous differentiation events. Thus, mTERT-MSC have been cultured onto PLA films, 30 and 100 microm PLA microbeads, and onto unpressed and pressed HYAFF-11 scaffolds. While the cells adhered preserving their morphology on PLA films, clusters of mTERT-MSC were detected on PLA beads and unpressed fibrous scaffolds. Finally, mTERT-MSC were not able to colonize the inner layers of pressed HYAFF-11. Nevertheless, such cell line displayed the ability to preserve Sca-1 expression and to retain multilineage potential when appropriately stimulated on all the scaffolds tested.

Effect of resveratrol on proliferation and telomerase activity of human colon cancer cells in vitro.

A number of studies performed in our laboratory and elsewhere, showed that resveratrol is able to prevent carcinogenesis and to impair tumor growth and progression. In order to provide additional information on the pleiotropic effects of resveratrol on malignant cells, the present study was performed to test the in vitro influence of the compound on the growth and TLMA of HT-29 and WiDr human colon cancer cell lines. The results confirmed that resveratrol has a direct, dose dependent, inhibitory effect on cell proliferation in both lines. In addition, for the first time, relatively high concentrations of this compound were found to be able to substantially down-regulate telomerase activity. These preliminary results further support the potential role of resveratrol in chemoprevention/chemotherapy of human colon tumor cells and provide the rational basis for novel strategies in cancer control.

O6-(4-bromothenyl)guanine (PaTrin-2), a novel inhibitor of O6-alkylguanine DNA alkyl-transferase, increases the inhibitory activity of temozolomide against human acute leukaemia cells in vitro.

Anti-tumour activity of triazene compounds of clinical interest [i.e. dacarbazine and temozolomide (TMZ)] relies mainly on the generation of methyl adducts to purine bases of DNA. Two DNA repair enzyme systems, i.e. the O6-guanine-alkyl-transferase (MGMT) and mismatch repair (MMR), play a predominant role in conditioning the cytotoxic effects of triazenes. In particular, high levels of MGMT associated with target cells are responsible of resistance to triazenes. On the contrary, the presence of MMR is required for the cytotoxic effects of these compounds. Previous studies performed by our group and a more recent clinical investigation reported by Karen Seiter, pointed out that triazene compounds could play an important role in the treatment of refractory acute leukaemia. Leukaemia blasts, especially of lymphoblastic leukaemia, show frequently high levels of MGMT activity. Therefore, it reasonable to hypothesize that combined treatment of leukaemia patients with triazene compounds along with MGMT inhibitors could lead to a better control of the disease. PaTrin-2 (O6-(4-bromothenyl)guanine, PAT) is a potent and scarcely toxic MGMT inhibitor recently introduced in clinical trials. This drug is used in combination with triazene compounds in order to augment their anti-tumour efficacy against neoplastic cells endowed with high MGMT activity. The present report describes, for the first time, pre-clinical in vitro studies on the cytotoxic activity of combined treatment with PAT+TMZ against long-term cultured leukaemia cells and primary leukaemia blasts obtained from patients with acute lymphoblastic leukaemia or acute myeloblastic leukaemia. The results point out that, both in long-term cultured leukaemia cell lines and in primary blast samples, PAT could improve dramatically the sensitivity of malignant cells to the cytotoxic effects of TMZ. This sensitizing effect is detectable when leukaemia cells show resistance mechanisms based on a MGMT-proficient phenotype. On the contrary, when resistance to TMZ is dependent on MMR deficiency, no influence of PAT can be detected in various experimental conditions. In conclusion, these results appear to provide disease-oriented rational basis to design novel clinical protocols for the treatment of acute leukaemia with combined administration of PAT and triazene compounds.

Focus on Fotemustine.

Fotemustine is a cytotoxic alkylating agent, belonging to the group of nitrosourea family. Its mechanism of action is similar to that of other nitrosoureas, characterized by a mono-functional/bi-functional alkylating activity. Worth of consideration is the finding that the presence of high levels of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT) in cancer cells confers drug resistance. In different clinical trials Fotemustine showed a remarkable antitumor activity as single agent, and in association with other antineoplastic compounds or treatment modalities. Moreover, its toxicity is generally considered acceptable. The drug has been employed in the treatment of metastatic melanoma, and, on the basis of its pharmacokinetic properties, in brain tumors, either primitive or metastatic. Moreover, Fotemustine shows pharmacodynamic properties similar to those of mono-functional alkylating compounds (e.g. DNA methylating drugs, such as Temozolomide), that have been recently considered for the management of acute refractory leukaemia. Therefore, it is reasonable to assume that this agent could be a good candidate to play a potential role in haematological malignancies.

Adjuvant therapy of melanoma patients (stage II, III): a pilot immuno-toxicological study.

A pilot study was conducted to assess the tolerability and the effect on host immunity of a post-surgery adjuvant treatment of melanoma patients with an anti-angiogenic agent, Tamoxifen (TAM, 20 mg/die p.o., daily), combined with immunomodulating cytokines, i.e. recombinant interleukin-2 (IL-2, 4 MUI/m2 s.c., day 8,10,12) and alpha-2b-interferon (IFN, 3 MUI/m2 i.m., day 15,17,19), starting a new cycle on day 21, for a total of 12 cycles. Fifty patients (pts) entered into the study, 27 males and 23 females with a median age of 55 years (range 25-75), performance status (ECOG) 0 with melanoma stage IIA (12 patients), stage IIB (28 patients), stage III (10 patients). Preliminary in vitro studies showed that TAM does not interfere with up-regulation of natural immunity induced by IFN, IL-2, or IFN + IL-2 in normal peripheral blood mononuclear cells (MNC). The clinical study indicates that the protocol was well tolerated. Increase of NK and LAK activity of patient MNC was observed on day 15. The mean disease-free interval was 10 months and 40 pts were alive at 5 years of follow-up. Further investigations should be performed to test effectiveness of this protocol in a randomized study.

Influence of Mycobacterium bovis bacillus Calmette Guérin on in vitro induction of CD1 molecules in human adherent mononuclear cells.

Nonpeptide antigens (including glycolipids of microbial origin) can be presented to T cells by CD1 molecules expressed on monocyte-derived dendritic cells. These HLA unrestricted responses appear to play a role in host immunity against Mycobacterium tuberculosis and other pathogenic bacteria. It is known that vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) has limited efficacy in many clinical settings, although the reasons for its inadequacy remain unclear. Here we have investigated the influence of BCG on the induction of CD1b on human monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF), which is believed to be the principal inducer of this antigen-presenting molecule. Although BCG alone led to a slight induction of CD1b expression, this agent reduced markedly the ability of GM-CSF to induce high levels of CD1b that were typically observed in uninfected cells. Inhibition of CD1b expression in BCG-infected monocytes was apparent at both the mRNA transcript and CD1b protein levels. Down-regulation of CD1b expression by BCG was mediated, at least in part, by one or more soluble factors and could not be reversed with high concentrations of GM-CSF or a variety of other cytokines. The present results suggest that BCG could diminish the efficiency of CD1-restricted T-cell responses against nonpeptide mycobacterial antigens by reducing CD1 expression on antigen-presenting cells. These findings have potential implications for understanding the nature of the immune response elicited by BCG in humans and suggest potential strategies that could be important for the development of better vaccines for the prevention of tuberculosis.

Saquinavir up-regulates telomerase activity in lymphocytes activated with monoclonal antibodies against CD3/CD28.

The present study describes the effect of the HIV protease inhibitor saquinavir on telomerase activity and interferon-gamma (IFN-gamma) production of nonadherent mononuclear cells (NA-MNC). Cells obtained from peripheral blood of healthy donors were exposed in vitro to a mixture of monoclonal antibodies against CD3 and CD28 membrane antigens in order to activate prevalently T cell subsets. Treatment with saquinavir was performed at the time of cell stimulation. Thereafter, NA-MNC were tested for telomerase activity (TRAP assay) and interferon-gamma production up to 7 days later. The results show that saquinavir up-regulates telomerase activity and IFN-gamma release in activated NA-MNC. These observations suggest that the anti-HIV effects of saquinavir could be accompanied by other immunopharmacological properties, influencing some aspects of the functional activity of immunocompetent cells. These include possible antagonistic effects against lymphocyte senescence, through telomerase activation, and a potentiating activity on the production of IFN-gamma following T cell activation.

Effect of Saquinavir on proliferation and telomerase activity of human peripheral blood mononuclear cells.

The present study describes the effect of Saquinavir on proliferation, interferon-gamma production and telomerase activity of non-stimulated, or activated non-adherent mononuclear cells (NAMNC), obtained from peripheral blood of healthy donors. Fresh NAMNC, non-stimulated or activated in vitro with PHA or with a mixture of monoclonal antibodies against CD3 and against CD28 membrane antigens (in order to obtain prevalent T cell responses), were exposed to Saquinavir before or at the time of mitogenic stimulation. Control and treated cells were tested for DNA synthesis (3H-thymidine incorporation), interferon-gamma production and telomerase activity (TRAP assay). The results indicate that Saquinavir is able to increase proliferation and interferon-gamma release in PHA-stimulated NAMNC, and telomerase activity either in non-stimulated and in PHA or antibody-activated cells. These results suggest that the activity against HIV infection afforded by Saquinavir, could be corroborated by its effects on the host. These include its adjuvant activity on mitogen-induced responses of lymphocytes, and its possible antagonistic effects against lymphoid cell senescence, through telomerase activation.

Poly (ADP-ribose) polymerase inhibitor increases apoptosis and reduces necrosis induced by a DNA minor groove binding methyl sulfonate ester.

The poly(ADP-ribose) polymerase (PARP) is involved in cell recovery from DNA damage, such as methylation of N3-adenine, that activates the base excision repair process. In the present study we demonstrated that MeOSO(2)(CH(2))(2)-lexitropsin (Me-Lex), a methylating agent that almost exclusively produces N3-methyladenine, induced different modalities of cell death in human leukemic cell lines, depending on the presence of PARP inhibitor. Growth inhibition, provoked by the combination of Me-Lex and PARP inhibitor, was associated with a marked down-regulation of c-myc, increased generation of single strand breaks and apoptosis. When used as single agent, at concentrations that saturated cell repair ability, Me-Lex induced mainly cell death by necrosis. Surprisingly, addition of a PARP inhibitor enhanced apoptosis and reduced the early appearance of necrosis. Telomerase activity was completely suppressed in cells exposed to Me-Lex alone, by 24 h after treatment, whereas it did not change when Me-Lex was combined with PARP inhibitor. Thereafter, inhibition of telomerase was observed with both treatments. The results suggest new insights on different modalities of cell death induced by high levels of N3-methyladenine per se, or by the methylated base in the presence of PARP inhibitor.

hMSH3 overexpression and cellular response to cytotoxic anticancer agents.

Mutations or transcriptional silencing of mismatch repair genes have been linked with tumour cell resistance to O(6)-guanine methylating agents, 6-thioguanine, cisplatin, doxorubicin and etoposide. Recently, it has been demonstrated that overexpression of the MSH3 protein is associated with depletion of the mismatch binding factor MutSalpha, and then with a marked reduction in the efficiency of base/base mismatch repair. In the present study we evaluated sensitivity of the HL-60 cell line and its methotrexate-resistant subline HL-60R, which overexpresses the hMSH3 gene, to a panel of chemotherapeutic agents. Cell growth inhibition induced by temozolomide, 6-thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine was significantly lower in the hMSH3-overexpressing HL-60R cell line as compared with the HL-60 parental line. Moreover, HL-60R cells were more resistant than HL-60 cells to chromosome aberrations induced by either N-methyl-N'-nitro-N-nitrosoguanidine or temozolomide, and to apoptosis triggered by the latter drug. Both cell lines were equally susceptible to growth inhibition induced by cisplatin, etoposide or doxorubicin. In addition, HL-60 and HL-60R cells showed comparable sensitivity to the clastogenic and apoptotic effects of cisplatin and etoposide. These results further confirm that loss of base/base mismatch repair is the most important molecular mechanism involved in cell resistance to O(6)-guanine methylating agents and 6-thioguanine. However, the status of the mismatch repair system could still influence tumour cell sensitivity to cisplatin, etoposide and doxorubicin, depending on the specific component of the system that is lost, and on the genetic background of the cell.

Combined effects of adenovirus-mediated wild-type p53 transduction, temozolomide and poly (ADP-ribose) polymerase inhibitor in mismatch repair deficient and non-proliferating tumor cells.

Lack of p53 or mismatch repair (MR) function and scarce cell proliferation are commonly associated with tumor cell resistance to antineoplastic agents. Recently, inhibition of poly(ADP-ribose) polymerase (PARP) has been considered as a tool to overcome resistance of MR-deficient tumors to methylating agents. In the present study we demonstrated that infection with p53 expressing adenovirus (Ad-p53), enhances chemosensitivity of MR-deficient tumor cell lines to the methylating agent temozolomide (TZM), either used as single agent or, more efficiently, when combined with PARP inhibitor. Moreover, the association of Ad-p53 with drug treatment induced a more pronounced growth inhibitory effect than that provoked by Ad-p53 infection only. Cells, growth arrested by p53 transduction, and then subsequently exposed to the drugs, were still highly susceptible to cytotoxicity induced by TZM and PARP inhibitor. The results suggested that this drug combination might be effective even in non-proliferating tumor cells. It is conceivable to envisage future possible strategies to enhance cytostatic or cytotoxic effects induced by Ad-p53, based on the use of TZM, alone or combined with PARP inhibitor for the therapy of resistant tumors.

Effects of single or split exposure of leukemic cells to temozolomide, combined with poly(ADP-ribose) polymerase inhibitors on cell growth, chromosomal aberrations and base excision repair components.

PURPOSE: To evaluate the antitumor activity of single versus split exposure of neoplastic cells to temozolomide (TZM) and poly(ADP-ribose) polymerase (PARP) inhibitor. METHODS: A leukemic Jurkat cell line and freshly isolated leukemic blasts were used. Jurkat cells are resistant to O6-methylguanine damage induced by TZM due to high levels of O6-alkylguanine-DNA alkyltransferase and to a functional defect in the mismatch repair system. Cells were treated with 3-aminobenzamide or with NU1025 to inhibit PARP activity. TZM was added to cell cultures immediately after PARP inhibitors. The concentrations of TZM used were 62.5 microM (corresponding to the peak plasma concentration in patients) or 125 microM. TREATMENT DESIGN: Cells were treated with 125 microM TZM plus PARP inhibitors (single exposure), or twice with 62.5 microM TZM plus PARP inhibitors with an interval of 24 h between treatments (split exposure). Tumor cell growth, clastogenicity and base excision repair gene transcripts or enzymatic activity were evaluated. RESULTS: The split exposure of Jurkat cells to TZM induced more pronounced and persistent growth inhibition and comparable chromosome damage in comparison with the single exposure. In addition, PARP inhibitors potentiated the cytotoxic effects induced by repeated treatment with TZM in fresh leukemic blasts. A marked decrease in X-ray repair cross-complementing 1 transcript and methylpurine glycosylase (MPG) transcript was detected in Jurkat cells subjected to the split exposure. In this case, a significant reduction in the corresponding enzymatic activity was also observed. CONCLUSIONS: Cytotoxicity induced by TZM and PARP inhibitors can be improved by a fractionated modality of drug treatment. The reduction in MPG transcript and function would presumably contribute to an increase in cell susceptibility to DNA damage induced by the methylating agent and PARP inhibitors.

Bacillus Calmette-Guerin down-regulates CD1b induction by granulocyte-macrophage colony stimulating factor in human peripheral blood monocytes.

Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 molecules to T cells appear to play an important role in host immunity against tuberculosis and other pathogenic bacteria. Since vaccination with Bacillus Calmette-Guerin (BCG) has limited efficacy, the influence of viable BCG organisms on the induction of CD1b antigen by granulocyte macrophage-colony stimulating factor (GM-CSF) has been tested in adherent mononuclear cells obtained from peripheral blood of healthy donors. The results indicate that the vaccine reduces substantially CD1b induction by GM-CSF. On the other hand, BCG was found to promote a slight increase in the expression of this molecule on target cells not exposed to GM-CSF. Attempts to reverse the antagonistic effects of BCG on GM-CSF with high concentrations of GM-CSF, alone, or associated with IL-4, were unsuccessful. Moreover, mycobacteria suppression by 10 microg/ml of rifampin, did not affect BCG influence on CD1b induction. The present results suggest that mycobacterium-induced impairment of the CD1 system could play a role in the unsatisfactory results obtained with BCG vaccination.

Effect of 5-fluorouracil on carcinoembryonic antigen expression and shedding at clonal level in colon cancer cells.

BACKGROUND: The carcinoembryonic antigen (CEA) is a tumor marker largely utilized for the detection of minimal disease or as a target of immunotherapeutic approaches. In preclinical models CEA has been found to be up-regulated after exposure of cancer cells to 5-fluorouracil (5-FU). In the present study, the clonal distribution of CEA and its regulation by 5-FU at clonal level was investigated using human HT-29 colon cancer cells. MATERIALS AND METHODS: The extent of CEA expression was measured in terms of: (a) antigen levels on plasma membrane, by flow cytometry; (b) cytoplasm and membrane protein, by Western blot analysis: (c) transcript, by Northern blot analysis; (d) CEA shedding by radioimmunossay. RESULTS: CEA protein and gene transcript were variably expressed among different clones. In all cases 5-FU was able to increase the percentage of CEA-positive cells, the amount of antigen, either in the membrane or cytosolic fractions, and the corresponding transcript. Moreover, a marked increase of CEA shedding was found in drug-treated cells with respect to that of controls. The increase of CEA induced by the antimetabolite was not the result of a selection mechanism based on preferential killing of CEA negative cells. The antimetabolite was capable of enhancing antigen expression also in other CEA-positive tumor cell lines with different basal levels of the marker. CONCLUSIONS: The present findings could be of potential value to increase the sensitivity of diagnostic procedures based on detection of CEA positive tumor cells. Moreover, the antimetabolite might be included in immunotherapeutic protocols to facilitate recognition of CEA-positive cancer cells by immune responses.

In vitro effect of hyperthermia on natural cell-mediated cytotoxicity.

It is well known that hyperthermia (HY), which is used for the treatment of cancer, depresses natural cell-mediated immunity in vitro. Experiments were performed to confirm the inhibitory effect of HY (42 degrees C for 1 hour) on natural killer (NK) activity and to evaluate the influence of HY on the generation and cytotoxic activity of interleukin-2 (IL-2)-activated NK cells. Additional experiments were also carried out to evaluate the effect of a simultaneous exposure of effector and target cells to HY. The results showed that HY profoundly reduced the lytic activity of NK cells and demonstrated that this inhibition was transient and not due to an apoptosis-induced reduction of the number of effector cells. Moreover, the exposure of mononuclear cells to HY before IL-2 stimulation did not affect the generation of IL-2-activated NK cells, whereas, the hyperthermic treatment of IL-2-activated NK cells produced a marked reduction of their cytotoxic activity. The results also showed that the simultaneous exposure of effector and target cells to HY, during the cytotoxicity assay, produced a marked reduction of lytic activity of NK and IL-2-activated NK cells, and that this impairment was specific for effector cells. In this context, heat-exposure of target cells alone, did not substantially modify their susceptibility to lysis induced by either NK or IL-2-activated NK cells. These results add further evidence of HY-induced inhibition of natural cell-mediated immunity, and suggest that, in the course of therapeutic HY, immune response could be significantly altered.