A WT1 antisense oligonucleotide inhibits proliferation and induces apoptosis in myeloid leukaemia cell lines.

The response of the CML-BC cell line, K562, the myelomonocytic cell line MM6 and the promyelocytic leukaemia cell line HL-60, to a 15 mer WT1 antisense oligonucleotide, targeted to the translation initiation site of the WT1 mRNA was examined. K562 cells exposed to 0.4 microM antisense oligonucleotide showed markedly reduced proliferation which was associated with reduced cell viability. Sense, scrambled and mutant antisense oligonucleotides had no effect on the proliferation of K562 cells. MM6 cells exposed to 0.4 microM antisense oligonucleotide also showed significantly reduced cellular proliferation which was also accompanied by loss of cell viability. In the K562 and MM6 antisense cultures that exhibited reduced cell viability, both DNA fragmentation and morphological features consistent with apoptosis could be identified. In contrast the growth of HL-60 cells was unaffected by exposure to 0.4 microM antisense oligonucleotide. In each of the cell lines examined, WT1 antisense oligonucleotide abrogated WT1 protein expression, and analysis of WT1 coding sequence in these cells showed that no oncogenic point mutations in the gene were present. We propose therefore that in some myeloid leukaemia cell lines, the expression of a normal WT1 protein is necessary for cell proliferation and that it plays a role in maintaining the viability of some leukaemia cells.

Trifluoroethanol and its oxidative metabolites: comparison of in vivo and in vitro effects in rat testis.

Trifluoroethanol (TFE) and trifluoroacetaldehyde (TFALD) produced a reduction in testis weight 3 days after a single oral dose of 10 mg/kg. In contrast, administration of trifluoroacetic acid (TFAA) caused no observable testicular effects. Reduction in testis weight was accompanied by morphological changes, involving specific damage to pachytene and dividing spermatocytes, and round spermatids. In an in vitro Sertoli/germ cell co-culture system, only TFALD was found to produce dose-related effects at concentrations of 10(-3) and 10(-4) M. There was increased germ cell loss from the cultures, particularly loss of pachytene and dividing spermatocytes, accompanied by leakage of the pachytene spermatocyte marker enzyme, lactate dehydrogenase-X. TFE and TFAA did not produce these effects in the culture system at concentrations equimolar with TFALD. These results suggest that TFALD may play a critical role in the development of the testis lesion seen with TFE in vivo. The effects seen both in vivo and in vitro were remarkably similar to those previously reported for another substituted alcohol and its metabolites, ethylene glycol monomethyl ether. It is postulated that the two series of compounds may have a similar mode of action on rat testis.

A comparison of the effects of the three isomers of dinitrobenzene on the testis in the rat.

Sexually mature Alpk/AP (Wistar derived) rats were killed 5 days after a single oral dose of 50 mg/kg of the 1,2-, 1,3-, or 1,4-isomers of dinitrobenzene. Testis weight reductions accompanied by testicular lesions were observed in the animals dosed with 1,3-dinitrobenzene (1,3-DNB) while the 1,2- and 1,4-isomers were without effect on the testis. However, 1,4-dinitrobenzene, but not 1,2-dinitrobenzene, was of a potency similar to that of 1,3-DNB in producing cyanosis and splenic enlargement in these animals, indicating that different mechanisms are probably responsible for these two toxic effects. In a subsequent study the pathogenesis of the testicular damage resulting from a single oral dose of 5, 10, 15, or 25 mg 1,3-DNB/kg was studied in sexually mature rats. Animals were killed 6, 12, 24, 48, and 96 hr after dosing and a detailed histopathological examination of the testes and selected tissues was made. At 12 hr after a single oral dose of 25 mg/kg, 1,3-DNB produced testicular lesions limited to Stages VIII to XI of the spermatogenic cycle. By 24 hr widespread Sertoli cell damage was evident and in some tubules was associated with degeneration of primary spermatocytes. Ultrastructural examination at this time confirmed that there were effects on Sertoli cells in the absence of germ cell damage. Similar effects were seen 48 hr after a single oral dose of 15 mg 1,3-DNB/kg. Doses of 5 or 10 mg 1,3-DNB/kg were without effect on the testis. The Sertoli cell is implicated as the prime target for the toxic action of 1,3-DNB with germ cell damage a secondary event.

Comparison of the in vivo and in vitro testicular effects produced by methoxy-, ethoxy- and N-butoxy acetic acids in the rat.

Methoxy-, ethoxy- and n-butoxy acetic acids, known urinary metabolites of the corresponding alkoxyethanol solvents, were administered by gavage to rats as a single oral dose equimolar with 500, 250 or 100 mg 2-methoxyethanol/kg body weight. Testicular weight and morphology were monitored over a 14-day period post-treatment. Methoxyacetic acid (MAA) was the only compound which produced a significant decrease in testicular weight. Histological examination of the testes from treated animals indicated that MAA at all doses and ethoxyacetic acid (EAA) at the highest dose, produced damage specific to spermatocytes undergoing meiotic maturation and division (particularly stages XIII-XIV) within 24 h of treatment. n-Butoxyacetic acid (BAA) had no discernable effect on the testis at any dose level or time, although there was evidence of haematuria produced by the compound. Addition of MAA, EAA and BAA to testicular cell cultures at concentrations approximately equivalent to the steady state plasma levels of MAA determined after a testicular toxic dose (500 mg/kg) of methoxyethanol (5 mM) produced a specific loss of pachytene spermatocytes (the target population in vivo) from the system by MAA and EAA (MAA greater than EAA). BAA did not produce any specific changes to testicular cell populations in vitro. Thus the production of testicular toxicity by alkoxyacetic acids diminishes with increasing chain length, and a good correlation exists between in vivo and the in vitro system.

Testicular toxicity of 2-methoxyacetaldehyde, a possible metabolite of ethylene glycol monomethyl ether, in the rat.

2-Methoxyacetaldehyde (MALD) was shown to produce specific cellular toxicity to pachytene spermatocytes in mixed testicular cell cultures as evidenced by morphological changes to these cells, an increase in germ-cell detachment and leakage of the pachytene spermatocyte marker enzyme lactate dehydrogenase-X. These effects occurred at concentrations where the known testicular toxicant, 2-methoxyacetic acid (MAA) was without effect (0.2 and 0.5 mM). In vivo, MALD also produced the characteristic testicular lesion reported previously for MAA and its parent compound ethylene glycol monomethyl ether (EGME). It is likely that MALD plays an important role in EGME-induced testicular toxicity.