Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology.

BACKGROUND: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors, and hematologic malignancies. The expression of isoforms of VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formation. METHODS: We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to beta(2)-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics. RESULTS: Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF(121) and VEGF(165) constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF(189) constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF(121) and VEGF(165) accounted for 94% (SD = 7.6%) and VEGF(189) only 6% (SD = 3.7%). CONCLUSIONS: Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.

Expression analysis and characterization of alternatively spliced transcripts of human IL-7Ralpha chain encoding two truncated receptor proteins in relapsed childhood all.

In the family of cytokines and cytokine receptors, alternative splicing of pre-mRNA is a frequently observed process that generates different protein isoforms from a single genetic locus. The splicing-derived cytokine receptor protein isoforms are mostly soluble receptors or show alterations in their cytoplasmic domain. It is possible that receptor abnormalities or a pathological ratio of different isoforms may contribute to leukaemia by circumventing normal growth factor control or altering the balance of proliferation and differentiation. IL-7 plays a critical role in early stages of both B and T cell maturation. Moreover, it stimulates the expansion of mature T cells including anti-tumour reactive cells as well as a number of T and B cell malignancies underlining its potential importance for deregulated lymphoid proliferation and leukaemogenesis. Here, we present detailed data on the expression of the interleukin 7 receptor alpha chain (IL-7Ralpha) in leukaemic cells from 210 children with acute lymphoblastic leukaemia (ALL) and describe two novel alternatively spliced transcripts of human IL-7Ralpha coding for truncated receptor proteins which are still capable of binding IL-7. IL-7Ralpha mRNA expression was more frequent in more mature pre-B ALL [91% (30/33)] than in common [81% (81/100)] or pro-B ALL [64% (18/28)], or even in T ALL [64% (29/45)]. These results are in concordance with flow cytometric analyses on the proportion of IL-7Ralpha bearing cells among total blast cell population. Our results lead us to assume that splicing derived IL-7Ralpha isoforms play a potential role in modulating IL-7 signal transduction and might be important for the pathogenesis of leukaemia.

Dose-response relationship of complementary radiotherapy following four cycles of combination chemotherapy in intermediate-stage Hodgkin's disease.

PURPOSE: To determine the appropriate irradiation dose after four cycles of modern combination chemotherapy in nonbulky involved field (IF/BF) and noninvolved extended-field (EF/IF) sites in patients with intermediate-stage Hodgkin's disease (HD). MATERIALS AND METHODS: HD patients in stage I to IIIA with a large mediastinal mass, E stage, or massive spleen involvement were treated with two double cycles of alternating cyclophosphamide, vincristine, procarbazine, and prednisone (COPP) plus doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by EF irradiation in two successive trials (HD1 and HD5). In the HD1 trial (1983 to 1988), 146 patients who responded to chemotherapy were randomized to receive 20 Gy (70 patients) or 40 Gy (76 patients) of EF irradiation in all fields outside bulky disease sites. A cohort of 111 patients who fulfilled the same inclusion criteria in the subsequent trial HD5 (1988 to 1993) were treated with 30 Gy. Bulky disease always received 40 Gy. RESULTS: Freedom-from-treatment-failure (FFTF) and survival (SV) curves showed no differences between the 20-, 30-, and 40-Gy groups. However, acute toxicities were more frequent in the 40-Gy arm. Analysis of relapse patterns showed that 18 of 26 relapsing patients either failed to respond in initial bulky sites (n = 5) or had an extranodal relapse (n = 9) or both (n = 4). After 5 years, the cumulative risk for relapse in bulky sites is 10%, despite 40 Gy of radiation. CONCLUSION: Our results strongly suggest that there is no relevant radiotherapy dose effect in the range between 20 Gy and 40 Gy in IF/BF and EF/IF after 4 months of modern polychemotherapy in patients with intermediate-stage HD. Relapse patterns indicate that patients destined to relapse need more systemic, rather than local, treatment. Based on our data, we conclude that 20 Gy is sufficient in EF/IF of intermediate-stage HD following four cycles of modern polychemotherapy.

Further chemotherapy versus low-dose involved-field radiotherapy as consolidation of complete remission after six cycles of alternating chemotherapy in patients with advance Hodgkin's disease. German Hodgkins' Study Group (GHSG).

OBJECTIVE: It was the aim of this prospective randomized multicenter study to compare chemotherapy and radiotherapy as consolidation treatments in patients achieving complete remission (CR) after 6 cycles of doxorubicin-containing chemotherapy in advanced-stage Hodgkin's disease (HD). METHODS: A total of 288 previously untreated patients aged 18-60 years with stage IIIB or IV HD received induction chemotherapy with 3 X (COPP + ABVD). Patients achieving CR were eligible for randomisation to either 20 Gy radiotherapy to initially involved fields (RT-arm) or to an additional 1 X (Copp + ABVD) (CT-arm ). Patients with nodal PR were allocated to more intense radiotherapy (IRT-arm: 20 Gy IF, 40 Gy to persisting tumor). Four patients with persisting organ involvement after induction received salvage chemotherapy. RESULTS: Of 288 patients, 171 (59%) achieved CR after induction chemotherapy. Of these, 100 patients were successfully randomized to RT or CT. In the CT arm relapses were observed on 10 of 49 patients compared with 13 of 51 patients in the RT arm (p = n.s.). Fifty patients refused randomisation and for them a treatment was chosen, and 21 patients refused any further treatment. Of these 21 patients with no consolidation therapy, 9 relapsed, indicating an approximately 3-fold increased relapse risk compared with those receiving either of the consolidation therapies. No relapse was observed in initially involved lung or liver sites. Adverse prognostic factors for freedom from treatment failure and survival were low hemoglobin and large mediastinal mass at initial presentation. CONCLUSIONS: No statistically significant differences in treatment efficacy were detected between 20 Gy IF radiotherapy and 1X (COPP + ABVD) chemotherapy following CR after six cycles of alternating chemotherapy in patients with advanced-stage HD. However, limited observations in a non-randomized cohort indicate that patients without consolidation treatment of CR after 6 cycles of chemotherapy may have an elevated risk of relapse.

Production of human adrenocorticotropin by cleavage of alkaline-phosphatase-derived fusion proteins containing repetitive recognition sequences for collagenases.

Recombinant plasmids coding for fusion proteins which consist of human adrenocorticotropin joined to N-terminal sequences of Escherichia coli alkaline phosphatase via collagenase-sensitive linkers were constructed and used for the production of these proteins by transformed E. coli cells. It was shown that repetitive linkers of the form -Gly-(Pro-Xaa-Gly)n-Pro- with n greater than or equal to 2 were cleaved by clostridiopeptidase A (Clostridium histolyticum) by orders of magnitude faster than corresponding nonrepetitive sequences (n = 1). The C-terminal cleavage product was Gly-Pro-adrenocorticotropin which could be converted to the authentic hormone by dipeptidyl peptidase IV. On the basis of these enzymatic reactions a procedure for the preparation of pure adrenocorticotropin was developed. Derivatives of alkaline phosphatase containing similar repetitive linker sequences were cleaved by clostridiopeptidase A as efficiently as the adrenocorticotropin fusion proteins.