[Acyclic nucleoside phosphonates as potential antineoplastic agents]. / Acyklické nukleosidfosfonáty jako potenciální antineoplastika.

Recently, Gilead Sciences (Foster City, CA, USA) presented a potential cytostatic drug GS-9219. It is a novel lipophilic prodrug of cyprPMEDAP, in vivo releasing the active compound PMEG in a two-step process. GS-9219 has shown a substantial therapeutic potential in treatment of spontaneous non-Hodgkin's lymphoma in dogs and its utilization in the human medicine is prospective. Hence, cyprPMEDAP represents a key intermediate in the intracellular activation of GS-9219. Both acyclic nucleoside phosphonates PMEG and cyprPMEDAP, serving as the basis for development of GS-9219, were discovered and their mechanism of action was investigated in detail at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. The biological studies using the rat lymphoma were carried out at the First Faculty of Medicine, Charles University.

Experimental therapy with 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP): origin of resistance.

The role of MRP4 and MRP5 transporters in the acyclic nucleoside phosphonate PMEDAP efflux was studied in vitro (CCRF-CEM cells) and in vivo (spontaneous transplantable T-cell lymphoma of SD/Cub inbred rats). The increased resistance against the cytostatic agent PMEDAP during longterm treatment was found to be associated with overexpression of MRP4 and MRP5 genes. The course of both gene activation differs significantly. While the MRP5 function is important in the onset of PMEDAP resistance, the intensity of the relative MRP4 gene expression increases rather continuously. Our data indicate cooperative acting of both MRP4 and MRP5 genes during the PMEDAP resistance development.

[Biological mechanisms of aging]. / Biologické mechanizmy stárnutí.

Contemporary views on the mechanisms of aging and aging variations--progeria and longevity are presented. Replicative aging, the roles of glycation and free radicals on the genetical determination of progeria and longevity are discussed.

Upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells.

L-Asparaginase is a standard component in chemotherapy of childhood acute lymphoblastic leukaemia (ALL). Leukaemic cells carrying TEL/AML1 fusion gene are more sensitive to treatment with L-asparaginase compared to other subtypes of ALL. We demonstrate in vitro the prolonged growth suppression of TEL/AML1[+] cells compared to TEL/AML1[-] leukaemic cells after L-asparaginase treatment simulating treatment protocol. Cell cycle analysis revealed TEL/AML1[+] cells to accumulate in G1/G0 phase (81-98%) compared to TEL/AML1[-] cells (47-60%). Quantitative analysis of asparagine synthetase (AsnS) expression showed the ability of TEL/AML1[+] cells to increase AsnS mRNA levels after L-asparaginase treatment to the same extent as TEL/AML1[-] leukaemic and nonleukaemic lymphoid cells. We hypothesise that TEL/AML1[+] cells are unable to progress into the S phase of cell cycle under nutrition stress caused by L-asparaginase, despite the ability of AsnS upregulation. Significantly higher expression of AsnS was found in untreated leukaemic cells from children with TEL/AML1[+] ALL (n=20) in comparison with the group of age-matched children with ALL bearing no known fusion gene (n=25; P=0.0043). Interestingly, none of the TEL/AML1[+] patients with high AsnS level relapsed, whereas 10/15 patients with AsnS below median relapsed (P=0.00028). Therefore, high AsnS levels in TEL/AML1[+] patients correlate with better prognosis, possibly reflecting the stretched metabolic demand of the lymphoblast.

Relevant animal model of human lymphoblastic leukaemia/lymphoma--spontaneous T-cell lymphomas in an inbred Sprague-Dawley rat strain (SD/Cub).

More than a decade of experimental work in an inbred subline of Sprague-Dawley rats having high incidence of spontaneous T-cell lymphoma/leukaemia is reviewed. Longitudinal follow-up of biological characteristics (growth, survival, haematology) of both multiple cases of primary disease and s.c. passaged lymphomas as well as comparative immunophenotypic and karyotypic studies are concluded. In these T-cell lymphomas (mostly CD4 positive), arising on the same genetic background of the inbred SD strain, the aberrations involving chromosome 11 have been recognized as a typical non-random cytogenetic marker. This unique rat model of lymphoblastic lymphomas/leukaemias, relevant to human pathology, seems to be very suitable for testing different anticancer therapeutic strategies, as it is documented by results of a number of various protocols conducted in our laboratory.

O-Phosphonatomethylcholine, its analogues, alkyl esters, and their biological activity.

O-Phosphonatomethylcholine, an isopolar phosphocholine analogue with a phosphonomethyl ether group replacing a phosphomonoester residue, was prepared by reaction of diisopropyl 2-chloroethoxymethylphosphonate with dimethylamine followed by quaternization of the thus-obtained diisopropyl 2-dimethylaminoethoxymethylphosphonate with iodomethane; the ester groups in the quaternary intermediate were cleaved with bromotrimethylsilane. Replacement of dimethylamine in the reaction sequence by morpholine and/or pyrrolidine gave the N-methylmorpholinium or N-methylpyrrolidinium analogues of O-phosphonatomethylcholine. Reaction of O-phosphonomethylcholine monotetrabutylammonium salt with 1-bromoalkanes in acetonitrile afforded a series of the corresponding monoalkyl (C10-C16) esters. None of these compounds except for the hexadecyl ester exhibited any appreciable cytostatic activity against DU-145, H460, HT-29, or MES-SA cell lines in vitro (evaluated by 3H-Thd incorporation assay). The hexadecyl ester exhibited modest in vitro cytotoxic activity comparable to that of the anticancer drug miltefosine (hexadecyl O-phosphocholine). In vivo evaluation of hexadecyl O-phosphonomethylcholine [transplanted SD lymphoma in inbred SD/cub rats, 10 mg kg(-1) day(-1) intratumoral injection for 10 days] resulted in a 40% decrease in lymphoma mass.

Antitumour activity of a combined treatment with PMEDAP and docetaxel in the Prague inbred Sprague-Dawley/cub rat strain bearing T-cell lymphoma.

Antitumour efficiency of combined therapy with N-9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) and docetaxel (DTX) was studied in an in vivo model of s.c. transplanted Sprague-Dawley (SD/Cub) rat T-cell lymphoma (phenotype SD10/96). The effect of the combined treatment of DTX with PMEDAP was significantly higher than that of DTX or PMEDAP alone. The s.c. administration of DXT into the vicinity of growing lymphoma together with i.p. administration of PMEDAP was found to be the most efficient combination. In this case, two out of four rats did not develop any lymphoma and remained alive. An irregular expression of Bcl2 protein was found in untreated and treated lymphomas, while the expression of protein p53 as well as MDM2 was not observed. All three types of the above-mentioned treatments (PMEDAP, DXT, DXT+PMEDAP) increased significantly the number of p21-positive cells, compared with untreated tumours.

Antitumour activity of N6-substituted PMEDAP derivatives against T-cell lymphoma.

The antitumour activity of four N6-substituted PMEDAP derivatives, Me2NEt-PMEDAP, allyl-PMEDAP, Me2-PMEDAP and cypr-PMEDAP, selected on the basis of their in vitro cytostatic activity, was studied in an in vivo model of haematological malignancy of inbred Sprague-Dawley rats. These compounds are believed to serve as the prodrugs of another (phosphonomethoxy)ethyl derivative, PMEG (9-[2-phosphonomethoxy) ethyl] guanine. We compared their toxicity and ability to inhibit tumour development in two different dosage regimes with those of their parent compound PMEDAP, as well with PMEG. The study confirmed the anticancer efficacy of the parental compound PMEDAP. Unlike PMEDAP, its N6-mono- and disubstituted congeners Me2NEt-PMEDAP, allyl-PMEDAP and Me2-PMEDAP were less potent or exhibited the same antineoplastic effect as PMEDAP. cypr-PMEDAP significantly decreased the survival of lymphoma-bearing rats due to high toxicity, which was approximately the same as that of PMEG. Therefore, these acyclic nucleoside phosphonates substituted at the 6-position of 2,6-diaminopurine ring do not seem to be promising drugs for the treatment of haematological malignancies.

Different mechanisms in inhibition of rat macrophage nitric oxide synthase expression by FK 506 and cyclosporin A.

The modulatory effect of FK 506 and cyclosporin A (CsA) on the expression of inducible nitric oxide synthase (iNOS) in macrophages and mechanisms of their action were analysed. Isolated rat peritoneal macrophages were cultured for 12 or 24 h with or without lipopolysaccharide (LPS) (5 microg/ml) and in the absence or presence of FK 506 or CsA (0.1 and 1 microg/ml). Total RNA from macrophages was isolated and the expression of the gene for iNOS was assessed by using RT-PCR. The concentration of NO2- in culture supernatants was taken as a measure of nitric oxide (NO) production. FK 506 (0.1 and 1 microg/ml) reduced the LPS-induced increase of NO2- levels by 68% and 81%, respectively. CsA (0.1 and 1 microg/ml) decreased levels of nitrites by 39% and 69%, respectively. The results obtained suggest that both immunosuppressive drugs exhibit dose-dependent inhibitory effect on NO production and that FK 506 is more potent agent than CsA, in this respect. FK 506 exhibits its inhibitory effect on a phosphatase at the transcriptional level in macrophages. iNOS expression down-regulation by CsA is occurred post-transcriptionally.

Anticancer effect of PMEDAP--monitoring of apoptosis.

Antitumor effect of N-9-[2-(phosphonomethoxy) ethyl]-2,6-diaminopurine (PMEDAP) was studied in an in vivo model of s.c. transplanted Sprague-Dawley (SD/cub) rat T-cell lymphomas. Three individual SD/cub neoplasias (SD10/96, SD14/97, SD1/90) of different phenotypes were used. During the treatment, survival of the rats, increase of lymphoma mass, and DNA fragmentation detected by APO/BRDU kit, as well as Bcl2 and p53 protein expression, were followed. The study gives evidence of the positive therapeutic effect of PMEDAP in two of the three tested lymphomas, SD10/96 and SD14/97. Slowly growing SD1/90 lymphoma differs from the others in a uniform karyotype with trisomy of chromosome 11, CD4- immunophenotype, heterogeneous cellular morphology and constitutive expression of p53 protein found in some neoplastic cells. Thus, the diverse anticancer efficacy of PMEDAP treatment among SD/cub lymphomas could be associated with the different phenotypes of individual neoplasias.