Tight regulation of the Src tyrosine kinase activity is essential for a variety of cellular processes, namely transitions of the cell cycle.
The peaks of Src activity are dependent on its posttranslational modifications as well as on the regulation of gene expression. The 3’UTRs of mRNAs are often crucial for rapid changes of the protein level. The chicken c-src 3’UTR effects on gene expression have been explored. The c-src 3’UTR decreased the in vivo tumorigenic potential of the src-activated mutants in chickens. This corresponds with the finding that the
c-src 3’UTR reduced the Src protein and src mRNA levels and luciferase activity in vitro. Our results suggest that the chicken c-src 3’UTR
plays a role in the negative control of gene expression, either transcriptionally or posttranscriptionally.
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Complex perioperative immunodysfunction occurs in patients with renal cell carcinoma undergoing nephrectomy. Here, the effect of pretreatment with IL-2 is addressed. Of 63 patients who underwent tumour nephrectomy, 26 patients received four doses of 10 Mio IE/m² IL-2 b.d. s.c. (i.e. a total of 40 Mio IE/m²) a week before operation, 37 did not. Parameters of cellular and humoral immunity (differential blood count, T-cell markers CD2, CD3, CD4, and CD8, B-cell markers CD19 and CD20, monocyte markers CD13 and CD14, NK-cell marker CD16, activation markers CD25, CD26, CD69 and HLA-DR, and cytokines IL-1-receptor antagonist (IL-1RA), IL-2, soluble IL-2-receptor (sIL-2R), IL-6, IL-10, and TGFß) were measured in peripheral venous blood. Blood was drawn before IL-2, one day before and immediately after the operation, and on the 1st, 3rd, 5th, and 10th postoperative days.
All patients showed postoperatively elevated leukocyte and granulocyte counts, and elevated serum levels of cytokines IL-6 and IL-10.
T-cell and activation markers were decreased. However, all these alterations were less accentuated in patients who had been pretreated with IL-2. Monocyte counts and IL-2 and TGFß levels were decreased, but IL-1RA and sIL-2R levels were elevated in pretreated patients. IL-2-related toxicity was WHO grade I-II in all patients, grade III in one patient. The anaesthetic regimen had no measurable effect. IL-6 concentrations were higher in renal venous than in venous pool blood, indicating IL-6 production in the tumour in vivo. Tumour-specific survival was better in pretreated patients with tumours extending beyond the kidney.
Pretreatment with IL-2 modulates perioperative immunodysfunction in patients undergoing tumour nephrectomy. This affects in particular T-cell-mediated immunity and levels of cytokines IL-10 and IL-6. The IL-2 application scheme used here was followed by distinct counter regulation including monocytes, IL-2, sIL-2R, IL-1RA and TGFß. Taken together, pretreatment with IL-2 may complement surgery in the treatment of patients with renal cell carcinoma, and may help close the therapeutic gap between neo-adjuvant and adjuvant immunotherapy.
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Adjuvant Autologous Tumour Cell-Lysate Vaccine versus No Adjuvant Treatment in Patients with M0 Renal
Cell Carcinoma after Radical Nephrectomy: 3-Year Interim Analysis of a German Multicentre Phase-III Trial
Ch. DOEHN, A. RICHTER, W. LEHMACHER, D. JOCHAM
Even M0 RCC is associated with tumour progression in approximately 30% of all patients after radical nephrectomy. Nevertheless, no effective adjuvant treatment after radical nephrectomy has been established. In a multicentre phase-III trial we investigated the impact of an adjuvant autologous tumour cell-lysate vaccination on the progression-free survival of patients with M0 RCC after radical nephrectomy.
Between January 1997 and August 1998 a total of 558 patients with a renal tumour were enrolled at 55 different centres (study group) in Germany. Prior to radical nephrectomy all patients were centrally randomized (Quintiles Germany) to either receive an adjuvant autologous tumour cell-lysate vaccine (6 applications at 4-week intervals after radical nephrectomy) or to receive no adjuvant treatment (control group) after radical nephrectomy. All patients were evaluated following standardized diagnostic investigations at 6-month intervals. Following the inclusion criteria (RCC stages pT2-3bpN0-3M0, TNM-classification, UICC 1993), 365 patients were evaluable for the 3-year progression-free survival analysis. There were 240 patients with stage pT2pN0M0 (104 in the vaccine group and 136 patients in the control group) and 89 patients with stage pT3pN0M0 (46 in the vaccine group and 43 patients in the control group). The remaining 36 patients had positive lymph nodes. The trial was performed according to ICH-GCP guidelines.
The 3-year progression-free survival rate for all tumour stages was 84.7% in the vaccine group and 80.9% in the control group. Patients with RCC stage pT3pN0-3M0 in the vaccine group demonstrated an advantage (74.4% in the vaccine group vs. 65.9% in the control group). For RCC stage pT2pN0-3M0 the 3-year progression-free survival rate in the vaccine group was 89.7.4% compared to 85.7% in the control group. Follow-up of all patients enrolled in this trial is ongoing.
This is the first randomized trial indicating a benefit from an adjuvant vaccination in patients with M0 RCC after radical nephrectomy. The advantage in terms of progression-free survival was more pronounced in patients with T3-tumours. However, it must be emphasized that the results of the final study report (2003) must be awaited before definite recommendations can be made.
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A Prospective Open-Label Single-Arm Phase II Study of Chimeric Monoclonal Antibody cG250 in Advanced
Renal Cell Carcinoma Patients
Z. VARGA, P. de MULDER, W. KRUIT, A. HEGELE, R. HOFMANN, C. LAMERS, S. WARNAAR, C. MALA,
S. ULLRICH, P. MULDERS
cG250 is an IgG1 kappa light-chain chimeric monoclonal antibody that binds to a cell surface antigen found on 95% of clear-cell renal cancer.
A multicentre phase II study was performed to evaluate the safety and efficacy of repeated doses of cG250.
Thirty-six patients with metastatic RCC were included. All patients were nephrectomized for the primary tumour. Twenty-one patients were pretreated (e.g. with IL-2, IFN-α). A weekly dose of 50 mg cG250 was given by iv infusion for 12 weeks. Patients with SD or tumour response (PR, CR) after 12 weeks of treatment could receive additional treatment for 8 more weeks.
None of the 36 enrolled patients had any cG250 grade III or IV toxicity. Only three patients had grade II toxicity possibly related to the study medication. ELISA testing gave no evidence for relevant amounts of HACA. Eleven patients presented with SD and ten were eligible for extension treatment. After the end of the study in the follow-up period, one patient demonstrated a CR in week 38 and another patient with SD showed a significant reduction of the overall tumour load in week 44. Six additional patients with progressive disease at study entry were stable for more than six months after the treatment start.
The weekly schedule of iv cG250 in patients with metastatic RCC was safe, very well tolerated and non-immunogenic in a 12-week treatment regimen. cG250 showed anti-tumour activity.
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Morphological symptoms of mesonephric kidney damage were analysed in chick embryos treated with nephrotoxic agents – CDDP or DBE.
The drugs were administered intraamniotically on ED 3 at doses 0.03 and 0.3 mg CDDP or 100 and 300 mg DBE per embryo. Body weight and absolute and relative measures of the mesonephroi (length, weight and form) were evaluated on ED 10. The higher doses of both agents affected the mass of this organ significantly. Simultaneously, a dose-dependent increase of renal malformations was detected in treated embryos, while the incidence of gross and cardiovascular defects was low (DBE) or absent (CDDP). Together with less pronounced effects on the total body growth, the results gave evidence for a higher sensitivity of the mesonephros to toxic insult when compared to
the whole organism. A direct cytotoxic effect multiplied by concomitant injury of blood supply seemed to be the main cause of CDDP nephrotoxicity. In the case of DBE, damage to the mesonephros was probably associated with a primary impairment of the vascular network. The chick embryo in ovo provides a promising system for the assessment of nephrotoxic effects induced by prospective therapeutic agents and environmental contaminants during the prenatal period.
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The variability in the morphology, modal number of chromosomes, TCR expression and functional reactivity of a CII-specific T-cell hybridoma at continuous subcultivation have been investigated. As the number of passages increased, besides the oval semiadherent cells (normal phenotype), fibroblast-like cells (transformed phenotype) were also observed. The two cell subpopulations differed in their karyotype characteristic, as well as in their functional reactivity. The cell population with a normal phenotype was characterized by a tetramodal number of chromosomes (30, 40, 48 and 70) and trisomies of chromosomes 6 and 14, while the cell population with a transformed phenotype was characterized by a trimodal number of chromosomes (11, 68 and 74) and trisomy of chromosome 12. A nullisomy of sex chromosomes was established in both types of cells. In the initial passages of subcultivation, 73.04% of the cells with a normal morphological phenotype expressed TCR-CD3 complexes on their surface and possessed high functional reactivity. After a two-week subcultivation, the values of these indices went down considerably: 46.11% of the cells expressed functional TCR-CD3 complexes, as a result of which their functional reactivity decreased. Only 2.71% of the cells with a transformed morphological phenotype expressed functional TCR-CD3 complexes on their surface. In these cells, a total loss of reactivity towards the specific antigens was established. The achieved results show that at continuous subcultivation the T-cell hybridomas are unstable, and with the increase in the number of passages there appear chromosome rearrangements, leading to loss of their functional reactivity.
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