Jan Buer

CXCR4/CXCL12 expression and signalling in kidney cancer.

CXCL12 (SDF-1), a CXC-chemokine, and its specific receptor, CXCR4, have recently been shown to be involved in tumourgenesis, proliferation and angiogenesis. Therefore, we analysed CXCL12α/CXCR4 expression and function in four human kidney cancer cell lines (A-498, CAKI-1, CAKI-2, HA-7), 10 freshly harvested human tumour samples and corresponding normal kidney tissue. While none of the analysed tumour cell lines expressed CXCL12α, A-498 cells were found to express CXCR4. More importantly, real-time RT–PCR analysis of 10 tumour samples and respective adjacent normal kidney tissue disclosed a distinct and divergent downregulation of CXCL12α and upregulation of CXCR4 in primary tumour tissue. To prove that the CXCR4 protein is functionally active, rhCXCL12α was investigated for its ability to induce changes of intracellular calcium levels in A-498 cells. Moreover, we used cDNA expression arrays to evaluate the biological influence of CXCL12α. Comparing gene expression profiles in rhCXCL12α stimulated vs unstimulated A-498 kidney cancer cells revealed specific regulation of 31 out of 1176 genes tested on a selected human cancer array, with a prominent stimulation of genes involved in cell-cycle regulation and apoptosis. The genetic changes reported here should provide new insights into the developmental paths leading to tumour progression and may also aid the design of new approaches to therapeutic intervention.

Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration.

After tissue loss the liver has the unique capacity to restore its mass by hepatocyte proliferation. Interleukin-6 (IL6)-deficient mice show a lack in DNA synthesis after partial hepatectomy (PH). To define better the role of IL6 and its family members for liver regeneration after PH, we used conditional knockout mice for glycoprotein 130 (gp130), the common signal transducer of all IL6 family members. We show that gp130-dependent pathways control Stat3 activation after PH. By using gene array analysis, we demonstrate that c-jun, NF-κB, c-myc, and tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the acute phase response was completely abolished, we considered that other means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and also IL6 −/− animals after PH leads to a significant reduction in survival and DNA synthesis, which was associated with decreased Bcl-xL expression and higher apoptosis in the liver. These results indicate that the phenotype concerning the reduction in DNA synthesis might be linked to the degree of infection after PH. Thus our results suggest that the role of gp130-dependent signaling is not a direct influence on cell cycle progression after partial hepatectomy but is to activate protective pathways important to enable hepatocyte proliferation.

Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells

Telomere shortening limits the regenerative capacity of primary cells in vitro by inducing cellular senescence characterized by a permanent growth arrest of cells with critically short telomeres. To test whether this in vitro model of cellular senescence applies to impaired organ regeneration induced by telomere shortening in vivo, we monitored liver regeneration after partial hepatectomy in telomerase‐deficient mice. Our study shows that telomere shortening is heterogeneous at the cellular level and inhibits a subpopulation of cells with critically short telomeres from entering the cell cycle. This subpopulation of cells with impaired proliferative capacity shows senescence‐associated β‐galactosidase activity, while organ regeneration is accomplished by cells with sufficient telomere reserves that are capable of additional rounds of cell division. This study provides experimental evidence for the existence of an in vivo process of cellular senescence induced by critical telomere shortening that has functional impact on organ regeneration.

Anaerobic adaptation in Pseudomonas aeruginosa: definition of the Anr and Dnr regulons

The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and biofilm formation during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network in response to oxygen tension and NO. Little is known about all members of the Anr and Dnr regulons and the mediated immediate response to oxygen depletion. Comprehensive transcriptome and bioinformatics analyses in combination with a limited proteome analyses were used for the investigation of the P. aeruginosa response to an immediate oxygen depletion and for definition of the corresponding Anr and Dnr regulons. We observed at first the activation of fermentative pathways for immediate energy generation followed by induction of alternative respiratory chains. A solid position weight matrix model was deduced from the experimentally identified Anr boxes and used for identification of 170 putative Anr boxes in potential P. aeruginosa promoter regions. The combination with the experimental data unambiguously identified 130 new members for the Anr and Dnr regulons. The basis for the understanding of two regulons of P. aeruginosa central to biofilm formation and infection is now defined.

Elevated pretreatment serum levels of soluble vascular cell adhesion molecule 1 and lactate dehydrogenase as predictors of survival in cutaneous metastatic malignant melanoma.

Very rapid progression of disease with a median survival of 6-9 months is a common feature of metastatic cutaneous malignant melanoma. Nevertheless, substantial variability of survival suggests that metastatic cutaneous malignant melanoma can be divided into several biological subgroups. Pretreatment serum levels of soluble adhesion molecules and various clinical parameters in cutaneous metastatic malignant melanoma were evaluated to determine their prognostic value. In this study pretreatment serum levels of soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble intercellular cell adhesion molecule 1 (sICAM-1), soluble endothelial leukocyte adhesion molecule 1 (sE-selectin) and multiple clinical factors were assessed in relation to overall survival of 97 consecutive patients with metastatic cutaneous malignant melanoma seen at our institution between May 1990 and April 1996. For statistical analysis, both univariate and multivariate Cox proportional-hazards models were used. Elevated pretreatment serum levels of sVCAM-1 (P < 0.005) and of lactate dehydrogenase (P < 0.002) were rendered statistically independent and were significantly associated with unfavourable outcome. Patients were assigned to one of three risk categories (low, intermediate and high) according to a cumulative risk score defined as the function of the sum of these two variables. There were significant differences in overall survival (P < 0.0001) between low- (n = 53, 5-year survival probability of 23.3%), intermediate- (n = 29, 5-year survival probability of 9.9%) and high-risk (n = 15) patients. Elevated pretreatment serum levels of sVCAM-1 and of lactate dehydrogenase correlate with poor outcome in metastatic cutaneous malignant melanoma. These data support risk stratification for future therapeutic trials and identify factors that need to be validated in prospective studies and may potentially influence decision-making in palliative management of patients with disseminated cutaneous malignant melanoma.

Interleukin 10 (IL-10) : an immunosuppressive factor and independent predictor in patients with metastatic renal cell carcinoma

SummaryInterleukin 10 (IL-10) is an immunosuppressive factor and has been detected in tumour cell cultures of renal cell carcinoma and of malignant melanoma. IL-10 has been described as a cytokine of the Th2 response; it is able to suppress antigen-presenting cells (APCs) and may lead to down-regulation of HLA class I and II molecules on dendritic cells and to anergy of T-lymphocytes. We evaluated pretreatment serum levels of soluble IL-10 and various clinical parameters to determine their prognostic value in 80 advanced renal cell carcinoma patients seen at our institution between May 1990 and April 1996. For statistical evaluation we used both univariate and multivariate Cox proportional hazards models. An elevated pretreatment serum level of IL-10 was a statistically independent predictor of unfavourable outcome (P < 0.0028), in addition to the well-known clinical and biochemical risk factors. These data support risk stratification for future therapeutic trials and identify a predictor which needs to be validated in prospective studies and may potentially influence decision making in palliative management of patients with metastatic renal cell carcinoma. These data also suggest a potential role of IL-10 in the development of advanced renal cell carcinoma and in the future design of therapeutic strategies.

Elevated serum levels of S100 and survival in metastatic malignant melanoma.

Current reports suggest serum S100 as a prognostic marker for disease progression in advanced malignant melanoma. In this study, we assessed serum levels of S100 and multiple clinical factors in relation to overall survival in 99 patients with metastatic malignant melanoma seen at our institution between May 1990 and April 1996. For statistical analysis, we used both univariate and multivariate Cox proportional-hazards models. Elevated serum levels of S100 correlated with poor outcome in metastatic malignant melanoma (P < 0.0001), univariate analysis). Upon multivariate analysis, however, S100 added no information to known clinical prognostic parameters.

Capecitabine in the treatment of metastatic renal cell carcinoma

To evaluate the therapeutic effects and systemic toxicities of a capecitabine-based home therapy regimen in patients with metastatic renal cell carcinoma, 30 patients were enrolled in a phase II clinical trial. Treatment consisted of oral capecitabine combined with subcutaneous recombinant human interferon-α 2a, recombinant human interleukin-2 and oral 13-cis-retinoic acid. There were two (7%) complete responses (CRs) and eight (27%) partial remissions (PRs), for an overall objective response rate of 34% (95% CI 17–53%). Except one, all responses are ongoing, with a median duration of 9+ and 8+ months for CRs and PRs, respectively. Additionally, 12 patients (40%) reached stable disease. Eight patients (27%) showed continued disease progression despite treatment. Therapy was well tolerated and was given in the outpatient setting. Capecitabine-related World Health Organization (WHO) grade 2 and 3 toxicities were observed in five and two patients respectively, and were limited to fatigue, nausea/vomiting, diarrhoea, stomatitis, dermatitis and hand-and-foot syndrome. The substitution of capecitabine for 5-FU in the pre-existing biochemotherapy regimen did not result in a reduced therapeutic efficacy and showed significant anti-tumour activity in patients with advanced renal cell carcinoma.

Biochemotherapy of advanced metastatic renal-cell carcinoma: results of the combination of interleukin-2, alpha-interferon, 5-fluorouracil, vinblastine, and 13-cis-retinoic acid.

SummaryWe conducted a phase I/II clinical trial evaluating the sequential outpatient combination of S.C. recombinant human interleukin-2 (rIL-2; given at 10 MIU/m2 b.i.d. on days 3–5 of weeks 1 and 4 and at 5 MIU/m2 on days 1, 3, and 5 of weeks 2 and 3), s.c. recombinant human alpha-interferon (rIFN-α; given at 6 MIU/m2 on day 1 of weeks 1 and 4 and on days 1, 3, and 5 of weeks 2 and 3 and at 9 MIU/m2 on days 1, 3, and 5 of weeks 5–8), i.v. bolus 5-fluorouracil (5-FU; given at 1,000 mg/m2 once weekly during weeks 5–8), and i.v. bolus vinblastine (given at 6 mg/m2 once weekly during weeks 5 and 8) in conjunction with p.o. 13-cis-retinoic acid (13-C-RA; given at 35 mg/m2 daily during weeks 1–8). Therapy was always given in the outpatient setting. Grade 3 constitutional symptoms (malaise, chills, fevers, anorexia) were observed in 4%–8% of treatment cycles and required a 50% reduction in the doses of rIL-2 and rIFN-α. None of the patients experienced major 5-FU-related toxicities such as severe diarrhea and/or stomatitis; up to 20% of patients developed vinblastine-associated peripheral polyneuropathy, which was reversible after the cessation of therapy. 13-cis-Retinoic acid produced no significant side effect; no toxic death occurred. Among 24 patients with progressive metastatic disease, there were 4 complete remissions (lung, lymph nodes) and 6 partial remissions (lung, pleura, liver, lymph nodes, and peritoneal carcinosis), for an overall objective response rate of 42% (95% confidence interval, 22%–63%). An additional 13 patients achieved disease stabilization (54%). The median time to response was 3–4 months (range, up to 6 months); all responses are continuous. In summary, although the potential synergy of biochemotherapy plus 13-cis-retinoic acid requires further preclinical investigation, the current outpatient combination regimen (rIL-2, rIFN-a, 5-FU, vinblastine, and 13-C-RA) proved to be both safe and highly effective in patients with advanced metastatic renal-cell carcinoma. A current multiinstitutional prospectively randomized trial is comparing biochemotherapy with and without concomitant 13-C-RA against rIFN-α plus vinblastine.

Prognostic impact of in vivo soluble cell adhesion molecules in metastatic renal cell carcinoma

SummaryThe purpose of the study was to determine prognostic significance of pretreatment serum levels of different molecules involved in cell to cell interactions along with other clinical parameters in patients with metastatic renal cell carcinoma. sICAM-1, sVCAM-1 and sELAM-1 serum levels were determined by ELISA assays in sera from 99 patients with histologically confirmed progressive metastatic renal cell carcinoma prior to initiation of systemic therapy. Kaplan–Meier survival analysis, log-rank statistics and two-proportional Cox regression analyses were employed to identify risk factors and to demonstrate statistical independence. In univariate analyses, the following pretreatment risk factors could be identified: serum sICAM-1 level > 360 ng ml–1, erythrocyte sedimentation rate > 70 mm h–1, serum C-reactive protein level > 8 mg l–1, serum lactic dehydrogenase level > 240 U/ l and neutrophil count > 6000 μl–1. Multivariate analyses demonstrated statistical independence for serum sICAM-1 level, erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) level as pretreatment predictors of overall patient survival. The prognostic significance of sICAM-1 might indicate a role of this molecule for tumour progression, potentially in association with the abrogation of anti-tumour immune responses. The possibility of defining a pretreatment risk model based on sICAM-1 level, ESR and CRP also warrants further investigation, with regard to a possible linkage between acute phase proteins and sICAM-1 levels.