9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP)--a potential drug against hematological malignancies--induces apoptosis.

Antitumor activity of the acyclic nucleotide analogs PMEDAP, PMEA, and PMEG was studied on a model of a spontaneous T-cell lymphoma in inbred SD/cub rats. Significant therapeutic effects were recorded after a treatment with 16 daily doses of PMEDAP at 5 mg/kg applied to the vicinity of the growing lymphoma. Identical administration of PMEA, or PMEG at a daily dose of 0.1 mg/kg did not affect the survival of lymphoma-bearing animals compared with untreated controls. A decrease in the lymphoma weight during PMEDAP administration was accompanied by the suppression of mitotic activity in neoplastic cells and increased chromatin condensation as witnessed by karyological examinations. Electron-microscopy showed the morphology of apoptotic cells (shrunken cells with condensed chromatin, apoptotic bodies) in lymphoma cell suspensions. An increase of nuclear DNA fragmentation was found during PMEDAP administration compared with spontaneous DNA fragmentation of untreated control lymphomas. These results indicate that PMEDAP application induces apoptosis in in vivo growing lymphomas. The antitumor effect of PMEDAP lasts only during the administration of the drug. After its cessation progression of neoplasia was reestablished.

G-banding chromosome studies of acute lymphoblastic Lewis rat leukemia (KPH-Lw-I).

Changes of karyotype in spontaneous acute lymphoblastic Lewis rat leukemia have been studied by conventional Giemsa staining method and by G- and C-banding techniques. Comparing with previously published normal findings in first passages on rats, an increasing number of breaks, gaps and fragments in the 5th and 14th passages has been proved. Chromosomal investigation performed after two-year transplantation of leukemia on syngenic animals revealed pseudodiploid karyotype of leukemic lymphoblasts with the persistence of cell line 42, XX, del 2, -7, -18, +2 mar, that remained unchanged up to the latest examination in April 1984. Remarkable stability of chromosomal changes such as del 2 and 7/18 translocation might indicate possible insertion sites of transforming (viral) agent and/or localization of putative oncogenes.

Spontaneous acute lymphoblastic leukemia in Sprague-Dawley rats. II. Cytogenetic analysis of nine individual leukemias.

Nine spontaneous acute lymphoblastic leukemias (SD1-SD9) in Sprague-Dawley rats were investigated cytogenetically by G-banding. The chromosome numbers of metaphase cells in all studied SD leukemias were near diploid and in all leukemias numerical or structural abnormalities were found. Affected were chromosomes No. 11, 2, 13 and 1. A common finding in most leukemias was trisomy 11 observed either as simple trisomy (SD1, SD4 and 2 clones of SD6), or the translocation form of trisomy (SD3), or the tandem translocation t(11;13) in SD7, or the Robertsonian fusion -rob(2;11) in SD5. In SD9 and one cell clone of SD8 no apparent trisomy was found, but the cells contained an 11q+ marker. Among the rearranged chromosomes, chromosome 2 was most frequently involved. Der No. 2 with terminal deletion was found in some of SD3 metaphases and a probable partial duplication of chromosome 2 was found in SD6. The finding of trisomy 2 in SD5 and SD6 occurred rarely. Further structural rearrangements concerned No. 13, the involvement of which was evident in the 13q+ marker typical of SD2, one cell clone of SD6, SD7 and SD8 leukemias. 1q+ aberration was the common finding in SD3 and SD5, while 1q-was observed in all SD6 metaphases. According to the cytogenetic examination of SD leukemias, we considered that the changes of chromosomes 11, 2, 13 and 1 were nonrandom. Based on the similarity of chromosomal rearrangements in individual leukemias, probable breakpoints on the affected chromosomes could be determined. 11cen, 11q12, 2q32-33, 2q16, 13q22, 1q43 and 1q54 were found to be the most frequently afflicted regions in SD leukemias.

Spontaneous acute lymphoblastic leukemia in Sprague-Dawley rats. I. Immunogenetic analysis of four individual leukemias.

The antigenic phenotyping of four individual spontaneous ALLs of SD strain revealed practically complete concordance of RT1 class I expression with PBL of SD strain. RT5 antigen as well as class II and SIg markers were not proven. T cell markers defined by MoAbs OX7 (Thy 1.1) and W3/13 (T cell common) were detected on all four ALL tested.

Antitumor activity of novel purine acyclic nucleotide analogs PMEA and PMEDAP.

PMEDAP and/or PMEA treatment of SD rat lymphomas significantly prolonged the mean survival time of tumor-bearing animals. Dose-dependent genotoxicity of both PMEDAP and PMEA was not observed in in vitro tests on stabilized diploid MRC-5 cell line. The mitotic activity of MRC-5 cells was completely inhibited after 48 hours exposure in culture medium containing PMEDAP (10 micrograms/ml), or PMEA (25 micrograms/ml), respectively. Significant concentration dependent inhibition of cell proliferation caused by PMEDAP and/or PMEA was also observed in murine splenocytes. The analogs specifically inhibit proliferation of mitogen-activated T-lymphocytes. Modulation of subpopulations of peripheral blood cells under in vivo conditions was found in inbred SD animals. Intraperitoneal administration of PMEDAP to young healthy SD animals induced the decrease of the CD4+/CD8+ value from 1.3-1.6 to 0.72 while i.p. application of PMEA caused a decrease of the same ratio to 0.62.

Chromosome 11 participation in acute lymphoblastic leukaemia in Sprague-Dawley rats.

Five cases of spontaneous acute lymphoblastic leukaemias (ALL) of Sprague-Dawley (SD) inbred rats have been studied cytogenetically. Chromosome studies were performed on direct preparations of the lymphomas using common cytogenetic methods. For detailed karyology, G-banded preparations were used. The chromosome numbers of metaphase cells in all leukaemias were near diploidy, the majority of metaphases being pseudodiploid with 1-2 stable marker chromosomes. The most frequent change involved chromosome 11, where the translocation form of trisomy 11 or 11q+ aberration was observed. Chromosome 11 abnormalities were found in all five leukaemias studied, suggesting to be a nonrandom change. 11 q11-q12 is supposed to be the critical region involved in the genesis or progression of SD ALL. These results are in agreement with our previous observations in nine cases of SD ALL (Sladká et al., 1988).

Cytogenetic changes in Sprague-Dawley rat lymphomas in the course of in vivo passages.

Changes in the karyotype in three spontaneous Sprague-Dawley rat lymphomas (SD7/95, SD8/96, SD9/96) have been studied in the course of in vivo passages. In individual lymphomas karyological findings of primary disease from lymph nodes were compared with changes found in the 1st and 10th passages of the lymphoma and bone marrow samples. Chromosome studies were performed on direct preparations using the G-banding technique. Chromosome counts of all specimens studied were near diploidy, the majority of metaphase cells being pseudodiploid. In later passages of two lymphomas, the tendency in selection to hyperdiploid karyotype, particularly in bone marrow was observed. The examination revealed an increased percentage of breaks in lymph node cells of primary disease and the existence of nonrandom change, derivative chromosome 11, which occurred in structural variability in all three lymphomas studied. The aberration involving chromosome 11 was evaluated as the addition of unknown material at chromosome band 11q11 or as a duplication or triplication of segment 11q12-q23. If this structural aberration was not found, the excessive derivative chromosome 11 or translocation t(11;13) was proved to be present. Further, rearrangements of chromosomes 13 and 7 were nonrandom chromosome abnormalities revealed in later passages of the lymphomas. The results are in accordance with our previous observations in 14 cases of SD lymphomas that showed nonrandom occurrence of rearrangements concerning chromosome 11 and also relatively frequent translocation involving chromosome 13.

Cytostatic effect of 9-(2-phosphonomethoxyethyl) adenine (PMEA). I. Lymphatic leukemia KHP-Lw-I in Lewis rats.

PMEA was administered i.p. daily on 10 consecutive days to inbred LEW rats inoculated with leukemic lymphoblastic cells KPH-Lw-I. The treatment was started 24 h after the inoculation. The observed cytostatic effects consisted in a significant prolongation of survival time associated with a suppression of the number of bone marrow leukemia cells with characteristic chromosomal marker of KPH-Lw-I leukemia as well as with a depression of the number of lymphoblasts in the blood.

Cytostatic effect of 9-(2-phosphonomethoxyethyl) adenine (PMEA). II. Lymphoblastic leukemia in Sprague-Dawley rats.

Two cases of spontaneous acute lymphoblastic leukemia in the inbred strain of Sprague-Dawley (Prague) rats have been observed. Since its reliable transplantability in syngeneic recipients was established, leukemic animals were used to test the cytostatic effect of PMEA. The treatment resulted in a significant prolongation of survival time of the treated animals. At the same time histological examination of PMEA-treated and untreated animals indicated that the drug effectively slows down the growth of lymphoma at the site of inoculation and inhibits the subsequent progression of tumor cells in the lung, liver, spleen and lymph nodes.

Antimitotic and teratogenic effects of acyclic nucleotide analogues 1-(S)-(3-hydroxy-2-phosphonomethoxyethyl)cytosine (HPMPC) and 9-(2-phosphonomethoxyethyl) adenine (PMEA).

The acyclic nucleotide analogues, HPMPC and PMEA, differ in their in vitro effect on the genetic material of eukaryotic cells. While HPMPC exerts a cytostatic effect on eukaryotic cells in vitro, PMEA has a genotoxic activity. These results correspond with the mode of embryotoxic action of these compounds: HPMPC exhibits a general embryolethal effect, whereas PMEA is apparently teratogenic and interacts with the mutant allele producing preaxial polydactyly of the hind limbs.

Chromosome assignment of Cd36 transgenes in two rat SHR lines by FISH and linkage mapping of transgenic insert in the SHR-TG19 line.

The chromosome position of the Cd36 insert was determined by FISH in two rat transgenic lines (SHR/Ola-TgN(EF1aCd36)10Ipcv (SHR-TG10) and SHR/Ola-TgN(EF1aCd36)19Ipcv (SHR-TG19). The Cd36 transgene construct labelled with digoxigenin-11-dNTP was used as a probe in the FISH analysis. In accord with the previous finding that the SHR-TG10 harbours 6-8 copies of the transgene, the signals from both metaphase and interphase nuclei of SHR-TG10 preparations were rather strong and the probe hybridized to both copies of chromosome 1 at band q55. The probe hybridization to SHR-TG19 metaphase preparations also showed homozygosity of the transgene with localization of both copies to chromosome 11 at band q11. The signals were distinct but much weaker compared to the SHR-TG10, which again is in accord with the fact that the SHR-TG19 line harbours only a single copy of the transgene. In order to look for a possible impact of the insertion site neighbourhood upon the transgene phenotypic effect, we performed linkage mapping of the transgene in the SHR-TG19 line. By linkage mapping, the placement of the transgene to the proximal part of RNO11 was confirmed, the critical interval being 4 cM between D11Rat20 and D11Rat21, in good agreement with the RH map. Within the close neighbourhood of the inserted Cd36 transgene, there are several genes known to be expressed in kidney, and so the influence of some regulatory sequences enhancing kidney expression of the Cd36 transgene can be envisaged.

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.

Genotoxicity of purine acyclic nucleotide analogs.

The genotoxic and embryotoxic effects of phosphonomethoxyalkylpurines, a new group of antiviral agents, decrease in the following order: PMEG > PMEthioG > PMEDAP > PMEA > (R)-PMPDAP = (R)-PMPA. Results of the present study are fully consistent with the previously found efficacy of their diphosphates to inhibit the replicative DNA polymerases. The marked genotoxicity of PMEG and PMEthioG is comparable to that of mitomycin C, whereas the moderate genotoxicity of PMEA is comparable to that of AZT. (R)-PMPDAP and (R)-PMPA did not induce any structural aberrations of chromosomes under the experimental conditions.