Jak3 expression and genomic sequence in pediatric acute lymphoblastic leukemia

Janus tyrosine kinase 3 (JAK3) is one of several key regulatory enzymes in B-cell precursors which is highly conserved between multiple species. The gene for Jak3 has been mapped to human chromosome 19p12-13.1 and encompasses 23 exons. Constitutively high levels of JAK3 activity may contribute to drug resistance and enhanced clonogenicity of leukemic B-cell precursors from children and infants with acute lymphoblastic leukemia (ALL). As part of a systematic effort to accurately determine the genomic sequence of Jak3 gene in normal and leukemic B-cell precursors, we sequenced a relatively short region of Jak3 spanning two introns, originally termed introns 10 and 11. This genomic sequence appeared in certain RT-PCR products from our analysis of Jak3 gene expression in pediatric, as well as infant, primary ALL cells. Unexpectedly, a gap in the original Jak3 genomic sequence was found in intron 10 across the sequence matching to an Alu element. Furthermore, the sequence obtained from intron 11 did not match at all to that previously reported, and the length of the intron was much larger than expected at 1.1 kb. Homology to Alu elements (three regions, 699 bp total) and a LINE2 element (one region, 189 bp total) were seen across the entire region covering exons 10-12 (2.1 kb total). Two potential single nucleotide polymorphisms (SNPs) were observed in intron 11. No apparent genomic mutation was found across this region in leukemic B-cell precursors from any of the ALL patients examined. This newly described sequence corrects the previous published genomic sequence from this region rather than identifying an insertion or translocation specific to these ALL cases. Our results significantly extend previous efforts to determine the genomic sequence of Jak3 and analyze its expression in childhood pro-B ALL and other forms of ALL.     

Ex vivo elimination of lymphoblastic leukemia cells from human marrow by mafosfamid

Studies were performed to evaluate the anti-tumor activity of mafosfamid, a new synthetic derivative of cyclophosphamide. We tested its ability to eliminate lymphoblastic leukemia cells from autologous bone marrow grafts following a 30 min preincubation in a highly sensitive clonogenic assay. Treatment with 50-100 micrograms mafosfamid/ml eliminated more than 4 logs of contaminating clonogenic tumor cells from a 200-fold excess of normal bone marrow. Flow cytometric studies showed differences in cell cycle kinetics between mafosfamid-resistant and mafosfamid-susceptible tumor cell clones. Compared to drug susceptible clonogenic tumor cells, clones that resisted treatment with 100 micrograms mafosfamid/ml exhibited a smaller percentage of cells in S-phase, indicating that mafosfamid is mostly cytotoxic to rapidly cycling tumor cells. The combination of mafosfamid and a target cell selective immunotoxin containing pokeweed anti-viral protein was superior to mafosfamid alone or immunotoxin alone for purging mafosfamid-resistant leukemic cells from human marrow.     

Reduced folate carrier and dihydrofolate reductase expression in acute lymphocytic leukemia may predict outcome: a Children's Cancer Group Study

PURPOSE: Methotrexate is a major component of current treatment regimens for children with acute lymphocytic leukemia (ALL). Potential mechanisms of methotrexate resistance include impaired drug uptake, decreased drug retention, and dihydrofolate reductase (DHFR) amplification. The purpose of this study was to assess whether reduced folate carrier (RFC) and DHFR expression in untreated leukemic blasts correlated with outcome. METHODS: Quantitative real-time RT-PCR was used to measure RFC and DHFR mRNA expression in leukemic blasts from 40 newly diagnosed patients with ALL obtained in a blinded fashion from Children's Cancer Group studies. RESULTS: Low RFC expression at diagnosis correlated significantly with an unfavorable event free survival. Surprisingly, low, not high, DHFR expression correlated significantly with an unfavorable event-free survival. Proliferative cell nuclear antigen (PCNA) expression demonstrated a weak inverse relationship between sample PCNA and DHFR or RFC expression, suggesting that DHFR and RFC expression may be markers for factors other than drug resistance. CONCLUSIONS: These results suggest that impaired transport may be an important mechanism of intrinsic methotrexate resistance in ALL, and DHFR expression also may be an important prognostic factor in ALL. Additional studies are necessary to clarify the mechanism for the correlation of low DHFR expression with poor outcome.     

Contraceptive activity of a spermicidal aryl phosphate derivative of bromo-methoxy-zidovudine (compound WHI-07) in rabbits

OBJECTIVE: To determine the vaginal contraceptive activity of WHI-07 in the rabbit model. DESIGN: Prospective, controlled study. SETTING: Center for advanced preclinical sciences. ANIMAL(S): Subgroups of 15, 16, or 24 New Zealand White does and 24 bucks per experiment. INTERVENTION(S): Ex vivo (Experiment 1) and in vivo (Experiments 2 and 3) treatment of semen with WHI-07 or Nonoxynol-9 (N-9). In Experiment I, ovulated does in subgroups of 15 were artificially inseminated with semen mixed with WHI-07 or vehicle. In Experiment 2, ovulated does in subgroups of 24 were artificially inseminated within 2 min after intravaginal administration of 2% WHI-07 gel-microemulsion or 2% N-9 gel and allowed to complete term pregnancy. In Experiment 3, ovulated does in subgroups of 16 were artificially inseminated at 15, 30, or 60 minutes. MAIN OUTCOME MEASURE(S): The numbers of implanted embryos on postinsemination day 8 or the proportion of does that became pregnant and delivered newborn rabbits; the litter size, weight, growth, and viability of pups until lactation day 5. RESULT(S): Exposure of semen to WHI-07 at the time of artificial insemination completely inhibited pregnancy rates (WHI-07-pretreated, 0%, vs. control, 60%) and embryo implantation (WHI-07-pretreated, 0/175 vs. control, 68/170). Intravaginal administration of a 2% WHI-07 gel-microemulsion or 2% N-9 gel before artificial insemination significantly inhibited pregnancy rates (81% and 85% inhibition, respectively) when compared with control. Furthermore, the 2% WHI-07 gel-microemulsion provided >90% inhibition of fertility even when insemination was delayed until 60 minutes after intravaginal application. Rabbits that delivered litters despite intravaginal application of 2% WHI-07 gel-microemulsion had healthy offsprings with no perinatal or postnatal repercussions. CONCLUSION(S): WHI-07 is a potent contraceptive agent in vivo. Intravaginal use of WHI-07 gel-microemulsion has clinical potential as a safe prophylactic contraceptive, in addition to its microbicide activity to curb the sexual transmission of HIV.     

Synthesis and anti-HIV activity of carbamates of antiviral agent stavudine

An efficient synthesis of carbamate analogues of the NRTI compound stavudine, has been achieved in five steps starting from commercially available thymidine. The synthesis involves conversion of thymidine into stavudine followed by condensation with carbaimidazole derivative obtained from various aromatic and heterocyclic amines in dimethylformamide solvent. The analogues thus obtained were further purified by crystallization to furnish analytically pure products. Examination of biological activity of these carbamate derivatives of stavudine showed that they inhibited HIV replication only at micro-molar concentrations.     

Prevention of islet allograft rejection in diabetic mice by targeting Janus Kinase 3 with 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (JANEX-1)

Janus kinase (JAK) 3-deficient mice were not able to reject allogeneic islet allografts. The JAK3 inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (CAS 202475-60-3, JANEX-1, WHI-P131) prevented the rejection of islet allografts in mice with a normal JAK3 expression status. The combination of JANEX-1 and cyclosporin A (CAS 59865-13-3) was more effective than either agent alone.     

High resolution X-ray structure of potent anti-HIV pokeweed antiviral protein-III

Pokeweed antiviral protein III (PAP-III), a naturally occurring protein isolated from late summer leaves of the pokeweed plant (Phytolacca americana), has potent anti-HIV activity by an as yet undetermined molecular mechanism. PAP-III belongs to a family of ribosome-inactivating proteins that catalytically deadenylate ribosomal and viral RNA. The chemical modification of PAP-III by reductive methylation of its lysine residues significantly improved the crystal quality for X-ray diffraction studies. Trigonal crystals of the modified PAP-III, with unit cell parameters a=b=80.47A, c=76.21A, were obtained using 30% PEG400 as the precipitant. These crystals contained one enzyme molecule per asymmetric unit and diffracted up to 1.5A, when exposed to a synchrotron source. Here we report the X-ray crystal structure of PAP-III at 1.6A resolution, which was solved by molecular replacement using the homology model of PAP-III as a search model. The fold typical of other ribosome-inactivating proteins is conserved, despite several differences on the surface and in the loop regions. Residues Tyr(69), Tyr(117), Glu(172), and Arg(175) are expected to define the active site of PAP-III. Molecular modeling studies of the interactions of PAP-III and PAP-I with a single-stranded RNA heptamer predicted a more potent anti-HIV activity for PAP-III due to its unique surface topology and more favorable charge distribution in its 20A-long RNA binding active center cleft. In accordance with the predictions of the modeling studies, PAP-III was more potent than PAP-I in depurinating HIV-1 RNA.