Treatment of refractory acute leukemia with timed sequential chemotherapy using topotecan followed by etoposide + mitoxantrone (T-EM) and correlation with topoisomerase II levels.

A phase I/II clinical study evaluated 17 patients with refractory/recurrent acute leukemia treated with 1.5 mg/m2/day topotecan on days 1-3 followed by etoposide (100 mg/m2/day)+mitoxantrone (10 mg/m2/day) on days 4, 5 and 9, 10. Timed sequential chemotherapy using the topoisomerase I-inhibitor topotecan before the topoisomerase II-inhibitors, etoposide+mitoxantrone (T-EM) treatment is proposed to induce topoisomerase II protein levels and potentiate the cytotoxic activity of the topoisomerase II-directed drugs. Fourteen patients had refractory and three had recurrent acute leukemia. The majority of patients were heavily pre-treated with greater than three re-induction chemotherapy regimens. Ten patients responded to T-EM treatment (59%). Four of seventeen (24%) had a complete remission and one had a partial remission. Four additional patients (24%) who scored complete leukemia clearance had no evidence of disease with complete white and red blood cell recovery but with platelet counts less than 100,000. The lack of platelet recovery in one patient having a partial response was scored as a partial leukemia clearance. The toxicity profile included major non-hematological toxicity including grade 3 mucositis (29%) and neutropenic fever (65%). Paired measurements of intracellular levels of topoisomerase II isoforms alpha and beta in leukemia blast cells (bone marrow) collected before (day 0) and after topotecan treatment (day 4) showed that a relative increase of topoisomerase IIalpha (Topo IIalpha) > or = 40% strongly correlated with response after T-EM treatment. Increased Topo IIalpha levels also corresponded to increased DNA fragmentation. Two patients who had an increase of Topo IIalpha of 20-25% had either a PR or PLC while patients with a < 10% increase showed no response to T-EM treatment. We conclude that timed sequential chemotherapy using topotecan followed by etoposide+mitoxantrone is an effective regimen for patients with refractory acute leukemia, and demonstrate Topo IIalpha protein level increases after topotecan treatment.

A 24 kDa parasitism-specific protein from the Caribbean fruit fly, Anastrepha suspensa: cDNA and deduced amino acid sequence.

A 24 kDa parasitism-specific protein (PSP24) was previously reported from the hemolymph of the Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae) after parasitization by the wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae). This study was designed to sequence the open reading frame of PSP24 and to determine whether it is encoded by the wasp, fruit fly host or by the entomopoxvirus D1EPV which is normally injected into the host with the wasp's egg. Utilizing an existing partial amino acid sequence of PSP24, we obtained two cDNAs by reverse transcription-polymerase chain reaction, from the host hemolymph 48 h post parasitization. The smaller cDNA has an open reading frame (ORF) that encodes 85 amino acids (aa) with a molecular mass of 9711.33 Da and the larger encodes 203 aa with a molecular mass of 23,076 Da. Both cDNAs share a common N-terminus with a signal peptide predictive of secreted proteins, a characteristic that agrees with the observed nature of PSP24. The mature proteins have 39 and 157 aa with deduced molecular masses of 4286.86 Da and 17,651 Da, respectively. Western blots of host hemolymph probed with the anti-PSP24 serum reveal proteins of 0.10 and 0.24 kD, respectively. The discrepancy between the deduced and the observed molecular masses may be explained by their predicted O-linked glycosylation. The amino acid sequences are not homologous with any protein in the available databases. Southern blot hybridization experiments revealed that the proteins are encoded by both the host and the parasite. Furthermore, injection of D1EPV into healthy fruit fly puparia induces the two proteins. Thus, in surprising contrast to an earlier hypothesis that D1EPV encodes PSP24, these results clearly demonstrate that the PSP24 proteins are encoded by wasp and fruit fly but not D1EPV genes. However, their expression is D1EPV induced.

P element excision in Drosophila is stimulated by gamma-irradiation in transient embryonic assays.

The influence of gamma-irradiation on P element excision and excision-site repair mechanisms was directly tested by embryonic somatic excision assays. Preblastoderm P[ry+, delta 2-3](99B) embryos, having a stable source of somatically active P transposase, were irradiated previous to injection with P excision indicator plasmids. Frequencies of precise or nearly precise P excision increased with gamma-ray doses ranging from 0.5 to 3.5 Gy. Higher doses resulted in frequencies close to that in unirradiated embryos, though considerable embryonic lethality was also evident at these doses. A direct positive interaction between gamma-irradiation and P element activity is concluded.

A Drosophila melanogaster hobo-white(+) vector mediates low frequency gene transfer in D. virilis with full interspecific white(+) complementation.

Transformation of a Drosophila virilis white mutant host strain was attempted using a hobo vector containing the D. melanogaster mini-white(+) cassette (H[w(+), hawN]) and an unmodified or heat shock regulated hobo transposase helper. Two transformant lines were recovered with the unmodified helper (HFL1), one containing only the white(+) marked vector, and a sibling line containing the vector as well as an HFL1 helper integration. An approximate total transformation frequency of 1% is deduced. A high frequency of wing and eye morphology mutants were also observed, suggesting that hobo may have mobilized a related element in D. virilis. The data reaffirms a relatively low transformation vector activity for the hobo transposon in D. virilis; however, nearly full interspecific expression white(+) marker supports its possible function in other species as well.

A new hobo, Ac, Tam3 transposable element, hopper, from Bactrocera dorsalis is distantly related to hobo and Ac.

A new transposable element from the hobo, Ac, Tam3 transposon family was isolated as a genomic clone from the oriental fruit fly, Bactrocera dorsalis. It is approximately 3.1 kb in length with 19-bp inverted terminal repeat sequences having a single mismatch. Though sharing several amino acid sequence identities with other hAT elements, it is distantly related to both hobo and Ac. Among hAT elements thus far described in insects, it is apparently the most distantly related to hobo.

The hobo transposable element excises and has related elements in tephritid species.

Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobo-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.

The hobo transposable element has transposase-dependent and -independent excision activity in drosophilid species.

Mobility of the hobo transposable element was determined for several strains of Drosophila melanogaster and several Drosophila species. Mobility was assessed by use of an in vivo transient assay in the soma of developing embryos, which monitored hobo excision from injected indicator plasmids. Excision was detected in a D. melanogaster strain (cn; ry42) devoid of endogenous hobo elements only after co-injection of a helper plasmid containing functional hobo transposase under either heat shock or normal promoter regulation. Excision was also detected in D. melanogaster without helper in strains known to contain genomic copies of hobo. In Drosophila species confirmed not to contain hobo, hobo excision occurred at significant rates both in the presence and absence of co-injected helper plasmid. In four of the seven species tested, excision frequencies were two- to fivefold lower in the presence of plasmid-borne hobo, hobo excision donor sites were sequenced in indicator plasmids extracted from D. melanogaster cn; ry42 and D. virilis embryos. In the presence of hobo transposase, the predominant excision sites were identical in both species, having breakpoints at the hobo termini with an inverted duplication of proximal insertion site DNA. However, in the absence of hobo transposase in D. virilis, excision breakpoints were apparently random and occurred distal to the hobo termini. The data indicate that hobo is capable of functioning in the soma during embryogenesis, and that its mobility is unrestricted in drosophilids. Furthermore, drosophilids not containing hobo are able to mobilize hobo, presumably by a hobo-related cross-mobilizing system. The cross-mobilizing system in D. virilis is not functionally identical to hobo with respect to excision sequence specificity.

Negative regulation of P element excision by the somatic product and terminal sequences of P in Drosophila melanogaster.

A transient in vivo P element excision assay was used to test the regulatory properties of putative repressor-encoding plasmids in Drosophila melanogaster embryos. The somatic expression of an unmodified transposase transcription unit under the control of a heat shock gene promoter (phs pi) effectively repressed P excision in a dose-dependent manner at very low concentrations relative to somatically active transposase (encoded by the hs pi delta 2-3 gene). Maximum repression required transcription of the complete transposase gene. Dose-dependent repression of P excision was also observed in the presence of a vector plasmid (pCarnegie4) having only the terminal sequences, including transposase binding sites, of the P element. However, repression required considerably higher concentrations of pCarnegie4 than phs pi, and elimination of P excision was not observed.

A functional analysis of the P-element gene-transfer vector in insects.

A P-element mobility excision assay was used to determine if non-drosophilid insects could support P gene vector function. Present studies included the testing of Muscids, Sphaerocerids, and Phorids, none of which were able to support P mobility. A new excision indicator plasmid was developed allowing the detection and recovery of virtually all P-element excision products. The frequency and sequence analysis of excision products from Drosophila melanogaster and another drosophilid, Chymomyza procnemis, indicated both quantitative and qualitative differences in the activity of transposase. The quantitative relationships observed in the original assay were maintained, and qualitative differences in transposase activity were reflected in the sequence of the empty donor sites. The results suggest that host factors are involved in cutting and ligating P-element DNA during excision, with transposase facilitating these processes. Possible limitations on P mobility by abnormal transposase transcript processing were tested in Anastrepha suspensa using transposase-encoding plasmids having deleted intron sequences. A transposase cDNA supported normal P excision in D. melanogaster, and a low level of mobility in A. suspensa. Possible applications of gene transfer in insects are presented, in particular methods to genetically sterilize and sex insects for the sterile-insect technique.

P element excision in Drosophila melanogaster and related drosophilids.

The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species. Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlike previous assays, this mobility assay permitted the recovery of excision products from plasmids regardless of whether the excision event was precise or imprecise. Both quantitative and qualitative differences between the products of excision in the various species studied were observed. The frequency with which P element excision products were recovered from D. melanogaster was 10-fold greater than from D. virilis and C. procnemis; however, the proportion of all excision events resulting in the reversion of a P-induced mutant phenotype was the same. Virtually all excision products recovered, including those resulting in a reversion of the mutant phenotype, did not result in the exact restoration of the original target sequence. Sequence analysis suggested that duplex cleavage at the 3' and 5' termini of the P element, or their subsequent modification, occurred asymmetrically and interdependently. P element-encoded transposase was not absolutely required for P element excision.