[Sites of action of vinca-alcaloids in protein and nucleic acid metabolism].
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Animals↗
Biomedical subjects
S Seeber
Publications and source records attributed to S Seeber.
Insertion of natural intron 6a-6b into a human cDNA-derived gene therapy vector for cystic fibrosis improves plasmid stability and permits facile RNA/DNA discrimination.
BACKGROUND: The gene therapy vector pCMV-CFTR containing human CFTR cDNA shows high segregational instability during growth in Escherichia coli. METHODS: By host strain screening and optimization of fermentation, satisfactory levels of pCMV-CFTR production were achieved. However, the vector was also vulnerable to structural instability manifested by the appearance during fermentation of a more stable mutant form in which the bacterial insertion sequence IS1 had transposed into exon 7 of plasmidborne CFTR. The instability of pCMV-CFTR is attributable to transcription from an upstream cryptic promoter leading to the production of CFTR peptide fragments known to be toxic when expressed in E. coli. To address this, we inserted the 1.1 kb natural human 6a-6b intron into pCMV-CFTR. RESULTS: The new vector pCMV-CFTR-int6ab is more stable in E. coli than either pCMV-CFTR or the IS1 mutant, grows to high cell density giving higher DNA yields and expresses CFTR appropriately in transfected cells. Thus, the intron has a stabilizing effect comparable to the IS1 insertion yet retains full functionality for gene therapy. We describe a PCR assay using primers directed to sequences flanking the intron that allows differentiation between DNA and mature mRNA. The T936C mutation present only in vector DNA has also been exploited to allow transgene CFTR to be distinguished and its dose-dependent expression to be detected in human cellular backgrounds. CONCLUSIONS: Instability of a plasmid vector for gene therapy has been minimized by rational modification. The introduction of an intron for this purpose offers the additional advantage of providing a discriminatory RT-PCR assay.
Amino Acid Sequence↗
Treatment of a cystic liver metastasis by intralesional injection of mitomycin C.
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Cysts↗
Prognostic impact of interferon alpha-induced cytogenetic remission in chronic myelogenous leukaemia: long-term follow-up.
To evaluate the long-term impact of the reduction of Philadelphia chromosome (Ph)-positive metaphases by treatment of chronic myelogenous leukaemia (CML) with interferon (IFN) alpha, we examined the outcome of 62 patients who had been enrolled between 1984 and 1990 into 2 IFN trials at our institution. As best cytogenetic response, 9 patients had achieved a complete remission and an additional 9 patients a partial remission. The remaining 44 patients had obtained either a minimal (n=29) or no cytogenetic response (n=15). Of the total of 62 patients, 9 were still on schedule and responsive to IFN in January 1995, including 7 patients in ongoing complete cytogenetic remission. The overall 5-year survival rate after a median follow-up from diagnosis of 51 months (range 3-102 months) was 62% and the median survival was reached at month 87. The effect of cytogenetic remission on survival was examined by "landmark" studies showing a significant survival advantage for patients with karyotype responses. In conclusion, in the patients studied, cytogenetic improvement was found to translate into improved survival expectancy. Long-term control by IFN alpha of CML, however, was restricted to a small minority of patients, predominantly to those attaining a complete suppression of the leukaemic cell clone as judged by cytogenetic criteria.
Actuarial Analysis↗
Sequential combination chemotherapy with vinblastine-bleomycin and doxorubicin-cis-dichlorodiammineplatinum(II) in disseminated nonseminomatous testicular cancer.
Forty-two patients with disseminated nonseminomatous testicular cancer were randomly entered to receive a two-armed chemotherapy regimen with mandatory crossover, consisting of either vinblastine-bleomycin or doxorubicin-cis-dichlorodiammineplatinum(II) (DDP) as initial therapy. Forty of these patients received at least four courses and were considered evaluable. Twenty-five of these patients received vinblastine-bleomycin and 15 received doxorubicin-DDP as initial treatment. The overall remission rate was 88%, with a complete remission rate of 68%. Thirty-two patients remain alive and 20 (50%) remain disease-free at 3+ to 18+ months, with a median duration of complete remissions of 9+ months. The response rate was not affected by prior chemotherapy or radiotherapy. Either treatment arm proved equally effective. However, response with doxorubicin-DDP therapy occurred in nine of 19 treatment courses when vinblastine-bleomycin therapy had failed, while response with vinblastine-bleomycin therapy occurred in only two of 11 treatment courses when doxorubicin-DDP therapy had failed. Thus, different patterns of cross-resistance between these alternative regimens may exist.
Adolescent↗
Chemosensitisation of alkylating agents by pentoxifylline, O6-benzylguanine and ethacrynic acid in haematological malignancies.
The effects of resistance modifiers (RM) on the cytotoxicity of mafosfamide (MAF), bis-chloroethylnitrosourea (BCNU) and dacarbazine (DTIC) were evaluated by the MTT colorimetric assay in isolated lymphocytes and blast cells derived from patients with chronic lymphatic leukaemia (CLL; n = 28) and acute myeloid leukaemia (AML; n = 30), or from healthy donors (n = 19). Pentoxifylline (PTX) has been shown to restore sensitivity to alkylating drugs by interfering with DNA repair. PTX (10 microM) significantly sensitised leukaemic blasts to the cytotoxic effect of MAF. In 8 out of 30 AML samples, sensitisation ratios (SRs; i.e. cytotoxic drug ID50s in the presence or absence of RM) for MAF in the presence of PTX were > 2 ranging up to 4.2. Inhibition of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) by O6-benzylguanine (O6-BG; 50 microM) enhanced the cytotoxicity of DTIC in CLL lymphocytes. SRs > 2 for DTIC in the presence of O6-BG were observed in 7 out of 28 CLL specimens. Sensitisation was generally greater in the more chemo-resistant specimens. Ethacrynic acid (EA; 1 microM), an inhibitor of glutathione-S-transferases (GST), failed to influence the cytotoxicity of alkylating agents in any cell type. Also, all examined RMs did not sensitive leukaemic cells to the cytotoxic effect of BCNU. The data show significant chemosensitisation of leukaemic cells to alkylating agents by PTX and O6-BG, indicating a potential clinical use of these substances as RM in patients.
Acute Disease↗