The influence of 2- chlorodeoxyadenosine in combination with tumour necrosis factor-α or its mutein on murine leukaemias L1210 and P388

We investigated the influence of 2-chlorodeoxyadenosine (2-CdA) in combination with tumour necrosis factor-α (TNFα) or its mutein VI, which differs from the native molecule by N-terminal amino acid composition, on the survival time of mice inoculated with leukaemia L1210 or P388. Groups of mice with leukaemia L1210 and P388 receiving 2-CdA combined with TNFα had shorter survival times than animals treated with these agents separately. In contrast, the administration of 2-CdA in conjunction with mutein VI, prolonged the survival of mice inoculated with these leukaemias as compared with animals receiving these agents separately. The results of the present study emphasize the importance of the biological activity of the TNFα molecule N-terminus.


Introduction
The combination of cytokines and cytotoxic drugs offers a new approach to increasing the therapeutic effect in the treatment of neoplastic diseases. There are many possible levels of interactions between these two groups of antineoplastic agents: cellular drug uptake, drug target enzymes, drug metabolizing enzymes.
2-Chlorodeoxyadenosine (2-CdA) is a new antineoplastic drug belonging to the purine analogues, which is especially active in lymphoproliferative disorders. Tumour necrosis factor-cz (TNF0t), a multimodal cytokine, has a wellknown antineoplastic activity. Muteins are TNFcz derivates, in which the first three to seven amino acids of the native molecule have been replaced. N-terminal amino acids play an important role in the binding of TNF to its cell surface receptors and in the biological activity of this cytokine.
The results of our previous experiments showed that both TNFcz and 2-CdA administered separately have therapeutic activity against murine leukaemias L1210 and P388. 2'3 Our recent studies have demonstrated that mutein VI has the best therapeutic activity against leukaemia P388. 4 The present study was designed to compare the influence of 2-CdA combined with TNF0t or with its mutein VI, on murine leukaemias L1210 and P388. DBA/2 strain, weighing 18-20g, 8-10 weeks old, purchased from the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (Wroclaw)were used. They were given standard laboratory food and water ad libitum.
Leukaemia: L1210 and P388 leukaemia cells were purchased from the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (Wroclaw) and were maintained by serial passages in the ascitic fluid of DBA/2 mice. Leukaemic cells from the fluid were resuspended in 0.9% sodium chloride so that 106 L1210 or P388 cells were injected i.p. into DBA/2 recipients. Therapeutics.. 2-CdA produced according to the method described by Kazimierczuk et aL 5 was kindly supplied by the Foundation of Development of Diagnosis and Therapy (Warsaw) as a dry powder. The synthesis and analysis of TNFz and its mutein VI were performed at the Department of Bioorganic Chemistry, L0d2, Poland. TNF had a specific activity of 2 x 107 U/mg. Mutein VI was constructed using synthetic oligonucleotides to induce changes in the cDNA encodin_ five Nterminal amino acids of native TNF-cz. Amino acid sequences were analysed by using automated Edman degradation with an Applied  Biosystems ABI 477A protein sequencer. The cDNA was expressed in E. coli and the resulting mutein was purified by ion exchange chromatography. Endotoxin contamination amounted to approximately 1  Statistical analysis: Statistical analysis was performed using Student's t-test. Results were considered significant when p < 0.05.

Results
TNF0t given once daily, at a dose 250 lig/kg, significantl prolonged the survival time of mice with leukaemia L1210. Nearly the same increase of lifespan in leukemia L1210-bearing mice was seen in the group receiving mutein VI in the sequential treatment schedule. Sequential application of TNF0t at a dose of 400btg/kg and daily application of mutein VI did not change the lifespan of leukaemia L1210-bearing mice as compared with animals of the control group (Table  1).

TNF in both treatment regimes and mutein VI
given daily at a dose of 250 gg/kg caused only a slight increase in the lifespan of P388 leukemiabearing mice. Sequential application of mutein VI at a dose 400 btg/kg significantly prolonged the survival time of mice with leukemia P388 ( Table  2).
The combination of 2-CdA and TNF shortened the survival time of animals in all treated groups. In contrast, mice receiving 2-CdA and mutein VI lived for a significantly longer time than did the animals treated with these agents separately (Tables 1 and 2).

Discussion
The interactions between cytokines and antineoplastic drugs are very complex. Cytokines can modulate the metabolism of the drug, the expression of adhesion receptors on tumour cells and gene expression. 7 Cytokines also have a great influence on the host's immune system. TNF is a polypeptide mediator of inflammation and cellular immune response. It also takes part in the induction of antineoplastic activity of the organism. The mechanism of TNF action is still unclear. Numerous different effects of this cytokine may be explained by both structural and functional heterogeneity of TNF receptors and by a diversification of post-receptor signal transduction pathways. 8 Although two cell surface receptors for TNF, p75 and p55, have been identified recently, the amino acid residues necessary for the biological activity of TNF are still not known. Klysik et al. constructed derivates of TNF termed 'muteins', in which the first three to seven amino acids of native TNF were replaced usin_ synthetic cDNA expressed in Eschechia coll.
The aim of our study was to compare the antineoplastic activity of native TNF + 2-CdA with that of mutein VI + 2-CdA. Our previous results have shown that among muteins III, V and VI, the latter has the greatest activity, against murine leukaemias L1210 and P388. Our results have shown that the combination of 2-CdA and TNF shortened the survival time of mice as compared with that of the control group. In contrast, mice receiving 2-CdA and mutein VI lived for a significantly longer time than did the animals treated with these agents separately. These results are in agreement with our previous observations that mice exhibit much better tolerance to high doses of mutein VI as compared with native TNF. 4 From other data '9 it is known that N-terminal amino acids are critical for both receptor binding and biological activity of TNF. Tch6rzewski et al. have revealed that mutein VI fails to recognize both TNF receptors. It also fails to increase secretion of cytokines IL-1, IL-6, GM-CSF and IFN-,, or to activate granulocytes, but it has the highest cytotoxic activity. '9 This may suggest an alternative mechanism of action.
TNF may cause tissue damage by augmenting neutrophil function, both by directly activating neutrophils and by affecting their response to other stimuli. 9 The lack of these effects after the addition of mutein VI may explain the lower toxicity when combined with 2-CdA as compared with the combination of native TNF and It is well known that TNF acts synergistically with topoisomerase target drugs (adriamycin, amsacrine, mitoxantron, etoposide, teniposide). This effect is probably related to the increase in the activity of topoisomerase I and II, resulting in enhanced DNA strand breaks and cleavage complex] In contrast, TNF does not enhance the antineoplastic effects of bleomycin, hydroxyurea, ara-C, cis-platin, mitomycin, vincristine and vinblastine. 7 Warzocha et al. have shown that TNF potentiates the antineoplastic activity of cyclophosphamide and methotrexate. 2 The studies of Winkelhake et al. have shown that the combination of TNFa and interleukin-2 does not increase the lifespan of L1210 leukaemia-bearing mice, compared with animals treated with these agents separately, m The results of our previous experiments revealed that 2-CdA and interferon-a show an additive inhibitory effect on the growth of clonogenic leukaemic blasts in vitro. 11 The mechanism of action of 2-CdA is still unclear, but it may be related to the activation of apoptosis. 12 Apoptosis (programmed cell death) represents a mechanism of cell removal in embryonic development and in controlling cell populations. Defects of this process may be involved in the production of neoplasms. Apoptosis may be induced by many factors, one of them being TNF. 13'14 TNF-activated protein kinase C appears to be involved in the induction of the c-jun, proto-oncogene which seems to be involved in apoptosis. 4--Induction of c-jun transcription is also observed in the treatment of myeloid leukaemia cells with new purine analogues, which have also been shown to induce apoptosis. 17 These observations may explain the synergistic action of 2-CdA and the TNF derivative, mutein VI.