The retinoblastoma protein (pRB) is an important regulator of development, proliferation, and cellular differentiation. pRB was recently shown to play a pivotal role in adipocyte differentiation, to ...interact physically with adipogenic CCAAT/enhancer-binding proteins (C/EBPs), and to positively regulate transactivation by C/EBPβ. We show that PPARγ-mediated transactivation is pRB-independent, and that ligand-induced transactivation by PPARγ1 present in RB+/+and RB−/− mouse embryo fibroblasts is sufficient to bypass the differentiation block imposed by the absence of pRB. The differentiated RB−/− cells accumulate lipid and express adipocyte markers, including C/EBPα and PPARγ2. Interestingly, adipose conversion of pRB-deficient cells occurs in the absence of compensatory up-regulations of the other pRB family members p107 and p130. RB+/+ as well asRB−/− cells efficiently exit from the cell cycle after completion of clonal expansion following stimulation with adipogenic inducers. We conclude that ligand-induced activation of endogenous PPARγ1 in mouse embryo fibroblasts is sufficient to initiate a transcriptional cascade resulting in induction of PPARγ2 and C/EBPα expression, withdrawal from the cell cycle, and terminal differentiation in the absence of a functional pRB.
To investigate the relationship between tumour hypoxia and serum and tumour osteopontin (OPN) levels.
Experiments were performed in CDF1 or C3H/Km mice implanted with a C3H mammary carcinoma (CDF1) ...or SCCVII squamous cell carcinoma (C3H/Km), respectively. Mice were either untreated or gassed with 10% oxygen for 1–72
h. Serum and tumour OPN levels were measured with an ELISA and tumour OPN mRNA levels using RT-PCR. Tumour oxygenation was estimated using the Eppendorf histograph with the percentage of
pO
2 values ⩽5
mm
Hg (HF5) as the endpoint.
OPN levels were 50-fold higher in the serum of non-tumour bearing CDF1 mice compared to C3H/Km mice. A tumour related increase in serum OPN levels was observed in CDF1 but not in C3H/Km mice. Low oxygen breathing increased HF5 in both tumour models and in the C3H mammary carcinoma model both serum and tumour OPN decreased after prolonged hypoxia (24
h and more). When 12
h of hypoxia was followed by 24
h reoxygenation there was a twofold increase in serum OPN levels. No changes were observed in the SCCVII model. No changes in tumour OPN mRNA expression were observed during hypoxia and reoxygenation in these tumour models.
Clear strain and tumour specific differences in the effect of hypoxia on OPN levels have been observed in two different mouse tumour models. These data emphasize the complexity in the relationship between poor oxygenation (and/or reoxygenation) of tumours and serum levels of OPN.
Identification of hypoxia regulated genes unaffected by pH Soerensen, Brita Singers; Horsman, Michael R; Overgaard, Jens ...
APMIS : acta pathologica, microbiologica et immunologica Scandinavica,
05/2008, Letnik:
116, Številka:
5
Journal Article
Recenzirano
Background. Genes upregulated by low oxygen have been suggested as endogenous markers for tumour hypoxia. Yet, most of the genes investigated have shown inconsistent results, which have led to ...concerns about their ability to be true hypoxia makers. Our previous studies have demonstrated that gene expression can be affected by factors other than oxygen concentration, e.g. extracellular pH (pHe). The current study is an in vitro approach, where we have used two different cell lines (SiHa and FaDuDD) under different conditions, and analysed gene expression with microarray to identify genes that are upregulated by hypoxia, independent of pHe. Methods. Human tumor cell lines uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma (FaDuDD) were used. Hypoxia was induced by gassing exponential growing cells in air tight chambers with various oxygen concentrations (21%, 5%, 1%, 0.1%, 0%) for 24 hours. Low pH media, titrated to pH 6.3 with a Tris-MES buffer system, was applied to some of the cells during the hypoxia treatment. RNA was extracted and gene expression was analysed using the Affymetrix - Human Genome U133 Plus 2.0 Array. Results. Using a multiclass SAM analysis combining both cell lines using four groups: normal oxygen - normal pH (21% and 5% oxygen at pH 7.4), low oxygen - normal pH (0.1% and 0% oxygen at pH 7.4), normal oxygen - low pH (21% and 5% oxygen at pH 6.3), low oxygen - low pH (0.1% and 0% oxygen at pH 6.3), we selected 1077 probesets (false discovery rate similar to 1%) that distinguish between the four groups. Amongst those probesets we could differentiate between genes induced at low pH, independent of oxygen concentration, genes induced at low oxygen only at normal pH, and genes induced at low oxygen at both normal and low pH. As our previous studies have shown, genes as Ca9 were found amongst the genes that was upregulated at low oxygen only at normal pH. 139 probesets, representing 96 different genes were found to be upregulated at low oxygen, independent of pH. Included in these 96 genes was Lysyl Oxidase, LOX, a recent suggested and promising hypoxia marker.
Objective:
Reverse transcription quantitative real-time polymerase chain reaction is efficient for quantification of gene expression, but the choice of reference genes is of paramount importance as ...it is essential for correct interpretation of data. This is complicated by the fact that the materials often available are routinely collected formalin-fixed, paraffin-embedded (FFPE) samples in which the mRNA is known to be highly degraded. The purpose of this study was to investigate 22 potential reference genes in sarcoma FFPE samples and to study the variation in expression level within different samples taken from the same tumor and between different histologic types.
Methods:
Twenty-nine patients treated for sarcoma were enrolled. The samples encompassed 82 (FFPE) specimens. Extraction of total RNA from 7-μm FFPE sections was performed using a fully automated, bead-base RNA isolation procedure, and 22 potential reference genes were analyzed by reverse transcription quantitative real-time polymerase chain reaction. The stability of the genes was analyzed by RealTime Statminer. The intrasamples variation and the interclass correlation coefficients were calculated. The linear regression model was used to calculate the degradation of the mRNA over time.
Results:
The quality of RNA was sufficient for analysis in 84% of the samples. Recommended reference genes differed with histologic types. However,
PPIA, SF3A1
, and
MRPL19
were stably expressed regardless of the histologic type included. The variation in ∆Cq value for samples from the same patients was similar to the variation between patients. It was possible to compensate for the time-dependent degradation of the mRNA when normalization was made using the selected reference genes.
Conclusion: PPIA, SF3A1
, and
MRPL19
are suitable reference genes for normalization in gene expression studies of FFPE samples from sarcoma regardless of the histology.
Reduction of the overall treatment time of radiotherapy increases the probability of local tumour control, but it does not benefit all patients. Identification of molecular marker profiles may aid in ...the selection of patients likely to benefit from accelerated radiotherapy.
Two hundred and nine patients with SCC of the supraglottic larynx received primary radiotherapy in the randomised DAHANCA trials to 66–68
Gy, 2
Gy/fx but with different overall treatment times of 9.5 week, 6.5 week and 5.5 week. Formalin-fixed paraffin embedded tumour slides were assessed by immunohistochemistry for expression of EGFr, E-cadherin, KI-67 and Bcl-2 and the
TP53 mutation profile was determined using PCR-amplification, DHPLC and sequencing. The profiles were established using a hierarchical clustering algorithm with a Bayesian information criterion for cluster number optimisation.
Full data-set were available for 158 patients and four almost equally sized clusters were identified. One of these clusters differed significantly with respect to local control compared to the other clusters: the cluster (
n=36) characterised by wild type
TP53, low expression of E-cadherin and Bcl-2, moderate KI-67 and EGFr, was not influenced by a reduction in the overall treatment time (
P=0.6) whereas the other clusters showed an increase in local control when the overall treatment time of radiotherapy was reduced. This was also partially seen with disease specific survival as the endpoint.
Molecular marker profiling may aid in the selection of patients that will benefit of a reduction in overall treatment time of radiotherapy in SCC of the supraglottic larynx.
Aim. To investigate the direct relationship between tumour hypoxia and lactate dehydrogenase (Ldh) levels in serum and tumour in two different pre-clinical murine models. Materials and methods. ...Experiments were performed in CDF1 or C3H/Km mice implanted with a C3H mammary carcinoma and SCCVII squamous cell carcinoma, respectively. Low oxygen breathing for 1-72 h was used to increase tumour hypoxia. Ldh activity was measured in the serum and tumour cytosole with a colorimetric method. Tumour Ldha mRNA levels were assessed with RT-PCR. Results. The serum Ldh in non-tumour bearing CDF1 mice and C3H/km mice was 10.5±2 U/ml and 12±2 U/ml, respectively. For C3H mammary carcinoma bearing mice, a positive correlation between tumour volume and tumour and serum Ldh was found. Tumour Ldh in SCCVII carcinomas also increased with increasing tumour volume, but no volume dependence of serum Ldh was found. Low oxygen breathing caused a 2-3 fold increase in tumour Ldha mRNA in both tumour models. In C3H mammary carcinoma bearing mice, serum and tumour Ldh significantly increased after 48 and 72 hours of hypoxia, respectively. Low oxygen breathing did not change serum and tumour Ldh in SCCVII carcinoma bearing mice. Reoxygenation for 4 or 24 hours had no additional effect on Ldh activity in any of the models. Discussion. Serum Ldh activity can be a marker for tumour burden in certain types of cancer. The relationship between serum and tumour Ldh and tumour hypoxia has not been confirmed. However, Ldha mRNA may be a potential new marker of tumour hypoxia and should be further investigated.
Abstract Antiprotons are interesting as a possible future modality in radiation therapy for the following reasons: When fast antiprotons penetrate matter, protons and antiprotons have near identical ...stopping powers and exhibit equal radiobiology well before the Bragg-peak. But when the antiprotons come to rest at the Bragg-peak, they annihilate, releasing almost 2 GeV per antiproton–proton annihilation. Most of this energy is carried away by energetic pions, but the Bragg-peak of the antiprotons is still locally augmented with ∼20–30 MeV per antiproton. Apart from the gain in physical dose, an increased relative biological effect also has been observed, which can be explained by the fact that some of the secondary particles from the antiproton annihilation exhibit high-LET properties. Finally, the weakly interacting energetic pions, which are leaving the target volume, may provide a real time feedback on the exact location of the annihilation peak. We have performed dosimetry experiments and investigated the radiobiological properties using the antiproton beam available at CERN, Geneva. Dosimetry experiments were carried out with ionization chambers, alanine pellets and radiochromic film. Radiobiological experiments were done with V79 WNRE Chinese hamster cells. The radiobiological experiments were repeated with protons and carbon ions at TRIUMF and GSI, respectively, for comparison. Several Monte Carlo particle transport codes were investigated and compared with our experimental data obtained at CERN. The code that matched our data best was used to generate a set of depth dose data at several energies, including secondary particle-energy spectra. This can be used as base data for a treatment planning software such as TRiP. Our findings from the CERN experiments indicate that the biological effect of antiprotons in the plateau region may be reduced by a factor of 4 for the same biological target dose in a spread-out Bragg-peak, when comparing with protons. The extension of TRiP to handle antiproton beams is currently in progress. This will enable us to perform planning studies, where the potential clinical consequences can be examined, and compared to those of other beam modalities such as protons, carbon ions, or IMRT photons.
A significant survival improvement after postmastectomy radiotherapy was identified in the Danish Breast Cancer Cooperative Group (DBCG82) b and c studies and in the British Columbia Randomized ...Radiation Trial. Recently, potential predictive value regarding response to postmastectomy radiotherapy was reported for carbonic anhydrase (CA) IX in a study (reported in abstract form) that included 160 patients. The purpose of the present study was to examine the importance of CA IX to response to postmastectomy radiotherapy in the larger scaled DBCG82 b and c studies.
The DBCG82 b and c studies included 3,083 high-risk Danish breast cancer patients. The women were randomly assigned to postmastectomy radiotherapy plus systemic therapy (cyclophosfamide, methotrexate and fluorouracil in premenopausal women; and tamoxifen in postmenopausal women) or to systemic therapy alone. Cores from invasive tumour-containing paraffin blocks from 1,000 patients (more than seven nodes surgically removed) were transferred to tissue microarrays. Tissue microarray sections were stained immunohistochemically for CA IX (M75). The median follow up for patients remaining alive was 17 years. Clinical end-points were loco-regional recurrence, distant metastases, disease-specific survival and overall survival. Statistical analyses included kappa statistics, chi2 or exact tests, Kaplan-Meier probability plots, Log-rank test and Cox regression analyses.
CA IX was assessable in 945 cores. The percentage of tumours positive for CA IX was 16% (> or = 10% invasive tumour staining). CA IX was not an independent prognostic marker for survival, distant metastases, or locoregional recurrence in the subgroup of 945 patients or within either of the two randomization arms. In subgroup analyses, however, CA IX was an independent prognostic marker for overall survival among postmenopausal women (P = 0.001), women with one to three positive nodes (P = 0.02) and hormone receptor positive women (P = 0.001). Fifteen-year probabilities of overall survival were improved by 9% and 7% after postmastectomy radiotherapy for the subgroups of CA IX negative and CA IX positive patients, respectively.
Within this series of 945 high-risk premenopausal and postmenopausal women, positivity for CA IX was not overall an independent prognostic marker for survival; only in subgroup analyses was it found to have prognostic value. The improvement in 15-year survival after postmastectomy radiotherapy was of similar magnitude in the two subgroups of CA IX positive and CA IX negative patients.
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