Influence of thymidylate synthase and p53 protein expression on clinical outcome in patients with colorectal cancer.

AIMS: Thymidylate synthase (TS) and tumor suppressor p53 are two proteins with an influence on tumor resistance to radio-chemotherapy that is well known. For this reason we tested the effect of TS and p53 expression on clinical outcome (tumor recurrence and survival) in patients after curative tumor resection, especially in patients who received adjuvant radio-chemotherapy. PATIENTS AND METHODS: A total of 120 patients with colorectal cancer were included in the study. A curative resection was possible in 83 patients, and 30 of this group received adjuvant therapy. For the immunohistochemical staining of tumor specimens, monoclonal antibody (mAb) TS 106 against TS and mAb DO-1 against p53 protein were used. TS positivity was defined as a moderate to high staining intensity in the cytoplasma of cells and p53 positivity as nuclear staining of tumor cells in >10% of these cells. RESULTS: Thymidylate synthase immunoreactivity was found in 59% of all cases and p53 staining in 51%. No relation between clinicopathological features and p53 expression was found in contrast to TS expression, where a highly significant association of TS-positive cases with tumor invasion (pT) was observed. Curatively resected patients with a TS-positive tumor developed tumor recurrence/distant metastases significantly more often than TS negative tumors. The same result was found when comparing p53-positive with p53-negative tumors and TS+/p53+ with TS-/p53- tumors. TS expression was highly significantly associated with poor survival and was the strongest independent prognostic factor in multivariate analysis, followed by lymph node status. CONCLUSION: Thymidylate synthase expression seems to be an independent prognostic factor and a possible predictor of tumor recurrence in patients with colorectal cancer.

Colorectal carcinomas with high MIB-1 labelling indices but low pKi67 mRNA levels correlate with better prognostic outcome.

AIMS: Antibodies specific to the proliferation-associated protein pKi67 (e.g. Ki67, MIB-1) are routinely used in oncology to assess the proliferation index of tumour cells. In untransformed cells the amount of pKi67 present at any time of the cell cycle is strictly regulated. To achieve a better understanding of expression and regulation of this protein in tumour cells, we investigated both pKi67 mRNA and protein expression in routinely fixed and embedded tissue of colorectal carcinoma. METHODS AND RESULTS: We determined a median pKi67 specific in-situ hybridization labelling index of 42% (9-79%) and a median Ki67 index (MIB-1 labelling index) of 59% (26-94%) in 47 resected colorectal adenocarcinomas of different stages and grades. In 32 cases expression of pKi67 mRNA and protein correlated well but we observed a significant difference between both values in 15 tumours. In these cases more than 30% of the cells expressed the protein but not the mRNA of pKi67, possibly due to cell cycle arrest. Patients belonging to this group had a significantly (P < 0.012) better prognosis. CONCLUSIONS: Tumours with a high pKi67 protein level but low mRNA expression are likely to proliferate more slowly than calculated on the basis of their Ki67 staining index, which possibly explains the patients' improved outcome.

[Influence of resterilized polypropylen meshes on growth of human fibroblasts--an experimental in vitro study]. / Einfluss resterilisierter Kunststoffnetze aus Polypropylen auf das Wachstum humaner Fibroblasten--Eine experimentelle in vitro Studie.

INTRODUCTION: We investigated the influence of resterilized polypropylen meshes (Prolene(R)) on proliferation and apoptosis of human fibroblasts in an experimental in vitro study. METHOD: Human fibroblasts were seeded into six-well culture dishes in a density of 3 x 10 (4) cells/well. After resterilization of meshes (steam autoclave, 121 degrees C, 20 min.) according to the manufacturer's recommendations (Ethicon, Norderstedt) square sheets of 2 x 2 cm were incubated with fibroblasts over a period of 6, 12, 18, 24, 30, 36, 42 and 48 h. Preparations of fibroblasts with non-resterilized meshes and without meshes served as controls. Proliferation index and apoptotic index were estimated by flow cytometry after cell staining with an FITC-conjugated antibody against the Ki-67 antigen or with FITC-conjugated Annexin-V and propidium jodide, respectively. RESULTS: A significant reduction of the proliferation index from 86 % to 42 % was found after 48 h incubation of cells with resterilized meshes, whereas only a slight decrease was found in the group with non-resterilized meshes (75 %) and in controls without meshes (80 %). Apoptotic index increased significantly from 2 % to 48 % after 48 h incubation with resterilized meshes in comparison to both control groups, where only a slight increase could be observed: non-resterilized meshes to 19 % and without meshes to 10 %. CONCLUSION: Resterilized meshes inhibit growth of human fibroblats in vitro significantly, demonstrated by a reduced proliferative activity and an increased apoptotic index. This could be caused by a release of toxic substances from the meshes, which have a negative influence on cell growth. Therefore, resterilization cannot be recommended.

[Polypropylene synthetic mesh modifies growth of human cells in vitro. An experimental study]. / Kunststoffnetze aus Polypropylen beeinflussen das Wachstum humaner Zellen in vitro. Eine experimentelle Studie.

INTRODUCTION: The aim of our study was to investigate the influence of polypropylene meshes on the proliferation and apoptosis of human cell cultures in vitro. METHODS: Human fibroblasts and HeLa cells were incubated in different densities (10(4), 3.10(4), 10(5) cells/well) together with polypropylene meshes (Prolene, 2 x 2 cm) in six-well culture dishes over a 48-h period. Cells without meshes served as controls. Cells were spun on slides and stained with the monoclonal antibody MIB-1. To calculate the proliferation indices, stained nuclei were counted. Apoptotic indices were determined by flow cytometric analysis, using FITC-conjugated Annexin-V and propidiumjodide for staining. RESULTS: Fibroblasts showed only a slight reduction of the proliferation index (PI) from 64% (controls) to 60% (meshes). Increasing cell density leads to a decrease in the PI of both groups. The PI of HeLa cells was similar in mesh groups and controls and independent of cell density. The apoptotic index (AI) of fibroblasts was significantly higher in the mesh group (3.7%) in comparison with the controls (1.9%). The same was observed for HeLa cells (AI mesh group: 4.5%, AI controls: 1.2%). Furthermore, an increase of AI was found with increasing cell density in both cell lines. CONCLUSION: Whereas meshes did not influence the proliferation of the cell lines examined, they seem to have a marked influence on apoptosis, as a significant increase of AI was observed in the mesh group in contrast to controls.

Assessment of proliferative activity in colorectal carcinomas by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).

The monoclonal antibody Ki-67 and the isospecific monoclonal antibody MIB-1 are routinely used in oncology to assess the proliferation index of tumor cells. A more objective and sensitive method is the determination of the of Ki-67 protein-specific mRNA by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In 25 resected colorectal adenocarcinomas of different stages and grades we determined between 0.2 and 4.4 amol (10(-18) mol) Ki-67 protein-specific mRNA per microgram total RNA (median = 0.88 amol). The corresponding Ki-67 indices (expressing the percentage of Ki-67/MIB-I positive tumor cells) ranged from 41 to 81% (median = 61%). We found a good correlation between Ki-67 index and mRNA expression (r = 0.75), a significant correlation between both data and tumor stage (primary tumor, regional nodes, metastasis [pTNM] staging classification) (p < 0.001), but not between both data and tumor grade. Both Ki-67 indices (p = 0.05) and mRNA levels (p = 0.014) correlated significantly to the patients' survival. These results demonstrate that the Ki-67 protein-specific quantitative RT-PCR is a useful method for the characterization of tumor cell proliferation.

p53 autoantibodies in sera of patients with a colorectal cancer and their association to p53 protein concentration and p53 immunohistochemistry in tumor tissue.

We evaluated p53 autoantibodies (p53-Ab) as a preoperative tumor marker and as a prognosis marker. We also investigated whether p53-Ab production is dependent on p53 protein overexpression in tumor tissue or on tumor volume. Serum samples of patients with a colorectal cancer (n = 130) and of healthy controls (n = 44) were examined for p53-Ab using an ELISA kit. P53 protein expression in tumor tissue was demonstrated immunohistochemically and quantified by ELISA. Tumor volume was calculated and patients' survival computed using the Kaplan-Meier method. p53-Ab were detected in the serum from 15% of patients; all controls were negative. There was a significant correlation between p53-Ab production and positive immunostaining or p53 protein concentration in tumor tissue. p53-Ab were detected at a higher percentage of patients with a tumor volume of 10 cm3 or greater than in those with a smaller tumor. No difference in patients' prognosis was found between the p53-Ab positive and negative groups. Because of their low sensitivity (15%) p53-Ab are not suitable as a preoperative tumor marker. However, their high specificity (100%) and their potential for early diagnosis of a tumor relapse makes them valuable for postoperative monitoring during follow-up in p53-Ab positive patients. Furthermore, their detection can be used as a simple serological test for early detection of p53 alterations.

Vascular endothelial growth factor (VEGF)--a valuable serum tumour marker in patients with colorectal cancer?

INTRODUCTION: Neo-angiogenesis, of great importance for tumour growth and nutrition, is preferentially mediated by the cytokine vascular endothelial growth factor (VEGF), which has a direct effect on vascular endothelial cell proliferation and migration. This study was designed to clarify whether VEGF is a suitable tumour marker in sera of patients with a colorectal cancer, and whether VEGF concentrations in sera and tumour tissues are correlated with tumour extension (pTNM) and especially with tumour volume or size. Furthermore, the influence of VEGF levels on patients >> prognosis was examined. METHODS: VEGF serum concentrations of 122 patients with colorectal cancer and 65 controls were determined with an ELISA kit. Additionally, VEGF concentrations of tumour and normal tissue were measured in 38 patients using the same ELISA. RESULTS: Our results demonstrate that VEGF is not a suitable diagnostic tumour marker in patients with colorectal cancer due to its low sensitivity (36%). However, a combination of the serum tumour markers CEA and VEGF can significantly increase the pre-operative diagnostic sensitivity to 62%. VEGF serum levels differed significantly between patients (mean 438 pg/ml) and controls (mean 203 pg/ml), and also between tumour and normal tissue (984 vs 89 pg/mg protein). Serum concentration showed a significant correlation to tumour volume and size. Patients with VEGF serum levels greater than cut-off had a poorer prognosis than those less than or equal to cut-off. For this reason VEGF could be used as a predictor of patients >> outcome.

Expression of p53 and mdm2 mRNA and protein in colorectal carcinomas.

The aim of our study was to investigate the expression of p53 and mdm2 mRNA and protein in colorectal adenocarcinoma. For the detection of mRNA, 60 fresh frozen human tumour samples and 12 samples of corresponding normal tissue were examined. After total RNA extraction, reverse transcription (RT) was performed followed by cDNA amplification with specific primers using RT-polymerase chain reaction (PCR). Immunohistochemical detection of protein was examined in 81 formalin-fixed and paraffin-embedded human tumour specimens as well as 15 samples of adjacent normal colorectal tissue. p53 mRNA was detected in 80% (48/60) of the tumours and in 67% (8/12) of normal tissue samples; 87% (52/60) of tumours had mdm2 mRNA in contrast to only 17% (2/12) of normal tissue specimens. Nuclear p53 protein expression was observed in 52% (42/81) of the tumour specimens and in none of the 15 normal specimens, whereas mdm2 protein was found in the nucleus (31%, 25/81) and also in the cytoplasm (86%, 70/81) of tumour samples. In normal tissue, mdm2 protein expression was only observed in the cytoplasm (13%, 2/15) and not in the nucleus. There was a significant correlation between coexpression of p53 and mdm2 protein and the occurrence of lymph node metastases (P = 0.03) as well as between p53 protein expression and the occurrence of distant metastases (P = 0.007). Additionally, significant associations were found between p53 mRNA and p53 protein, p53 mRNA and mdm2 mRNA or protein, and also between mdm2 mRNA and mdm2 protein expression, supporting the existence of a regulatory mechanism involving p53 and mdm2.

[Immunocytochemical and molecular proof of disseminated tumor cells in bone marrow, peritoneal lavage, blood and regional lymph nodes. A review of the literature and own results]. / Immunzytochemischer und molekularer Nachweis disseminierter Tumorzellen im Knochenmark, der Peritoneallavage, dem Blut und den regionären Lymphknoten. Literaturübersicht und eigene Ergebnisse.

Immunocytochemical or molecular detection of disseminated tumor cells in different compartments (bone marrow, abdomen, venous blood, lymph nodes) is becoming more and more important in determining the complete extent of the tumor at the time of primary therapy. However, their prognostic relevance is not clear up to now. Whereas their appearance in bone marrow revealed a poor prognosis in some studies, contradictory results have been obtained from examinations of lymph nodes. Moreover peritoneal lavage and venous blood have seldom been examined with these methods. Because standardization of the immunocytochemical or molecular techniques does not exist, therapeutical conclusions can not be drawn from their detection at this time.

Protection of photosynthesis against ultraviolet-B radiation by carotenoids in transformants of the cyanobacterium synechococcus PCC7942

The cyanobacterium Synechococcus PCC7942 was transformed with various carotenogenic genes, and the resulting transformants either accumulated higher amounts of beta-carotene and zeaxanthin or showed a shift in the carotenoid pattern toward the formation of zeaxanthin. These transformants were exposed to ultraviolet-B (UV-B) radiation, and the degradation of phycobilins, the inactivation of photosynthetic oxygen evolution, and the activity of photosystem II were determined. In the genetically modified cells, the influence on destruction of phycobilins was negligible. However, protection of photosynthetic reactions against UV-B damage was observed and was dependent on the carotenoid concentrations in the different transformants. Furthermore, it was shown that endogenous zeaxanthin is more effective than beta-carotene. Our results suggest that carotenoids exert their protective function as antioxidants to inactivate UV-B-induced radicals in the photosynthetic membrane.

Expression of MDR1 mRNA and encoding P-glycoprotein in archival formalin-fixed paraffin-embedded gall bladder cancer tissues.

The aim of this study was to examine the expression of P-glycoprotein (Pgp) and MDR1 mRNA, in gall bladder carcinoma, a chemo-resistant tumour. 26 cases of gall bladder cancer and nine samples of normal gall bladder archival paraffin blocks were investigated for the presence of Pgp protein with immunohistochemistry (IHC) and MDR1 RNA by reverse transcription-polymerase chain reaction (RT-PCR). Monoclonal antibodies JSB-1 and UIC-2, recognising separate epitopes of Pgp, were used for IHC. For RT-PCR, total RNA was extracted from paraffin-embedded tissue. After RT, the samples were subjected to nested PCR (NPCR) using primers specific for the MDR1 gene, and evaluated by electrophoresis. In gall bladder carcinoma, the percentage of positive cases expressing Pgp (77% for JSB-1, 69% for UIC-2) and MDR1 mRNA (52%) was significantly higher than those in normal gall bladder. In earlier TNM stages Pgp and MDR1 mRNA were more frequently expressed (non-significant) than in advanced stages. The results of this study suggested that overexpression of MDR1 mRNA and Pgp in gall bladder carcinoma tissue probably is a very important reason why gall bladder cancer is generally not responsive to chemotherapy.

[Determination of vascular endothelial growth factor (VEGF) concentration in serum of patients with colorectal carcinoma]. / Bestimmung der Vascular Endothelial Growth Factor (VEGF)-Konzentration im Serum von Patienten mit einem kolorektalen Karzinom.

The aim of our study was to determine the serum concentration of VEGF in 53 patients with a colorectal carcinoma and 22 healthy volunteers (control group) and to compare it with tumor stage and volume. We found significantly higher serum levels in tumor patients in contrast to the control group and between patients with and without distant metastases. There was also a correlation between high serum concentrations and great tumor volumes, but only weak with tumor stage. Our results support the hypothesis that tumor growth is dependent on angiogenesis and that VEGF is a potent growth factor with angiogenic activity.

Expression of Erwinia uredovora Phytoene Desaturase in Synechococcus PCC7942 Leading to Resistance against a Bleaching Herbicide.

The gene coding for phytoene desaturase of the bacterium Erwinia uredovora (crtI) was inserted into the chromosome of the cyanobacterium Synechococcus PCC7942 strain R2-PIM8. For expression of crtI in the heterologous host, two constructs with different promoters were introduced into Synechococcus. In the first, crtI was fused to the 5[prime] region of the psbA gene of the xanthophycean microalga Bumilleriopsis filiformis. The second construct carried crtI inserted downstream of the neomycin phosphotransferase II gene (nptII) from the transposon Tn5. Expression of crtI under the control of the respective promoter was shown by immunodetection of the gene product. The functionality of the heterologously expressed phytoene desaturase CRTI in the transformants was demonstrated by enzymic assays. The transformants acquired very strong resistance toward the bleaching herbicide norflurazon.

Identification of cfxR, an activator gene of autotrophic CO2 fixation in Alcaligenes eutrophus.

A regulatory gene, cfxR, involved in the carbon dioxide assimilation of Alcaligenes eutrophus H16 has been characterized through the analysis of mutants. The function of cfxR is required for the expression of two cfx operons that comprise structural genes encoding Calvin cycle enzymes. CfxR (34.8 kDa) corresponds with an open reading frame of 954 bp, with a translational initiation codon 167 bp upstream of the chromosomal cfx operon. The cfx operon and cfxR are transcribed divergently. The N-terminal sequence of CfxR is very similar to those of bacterial regulatory proteins belonging to the LysR family. Heterologous expression of cfxR in Escherichia coli was achieved using the pT7-7 system. Mobility shift experiments demonstrated that CfxR is a DNA-binding protein with a target site upstream of both the chromosomal and the plasmid-encoded cfx operons.

On the operon structure of the cfx gene clusters in Alcaligenes eutrophus.

Three transposon Tn5-induced mutants deficient in autotrophic CO2 fixation were isolated from a megaplasmid pHG1-cured strain of Alcaligenes eutrophus H16. Their phenotypes were initially characterized by their ability to form both key enzymes of the Calvin cycle, ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoribulokinase (PRK). Since the transposon insertions were at different sites within the chromosomal cluster of cfx genes encoding Calvin cycle enzymes, the individual mutants showed different inactivation patterns for Rubisco and PRK synthesis. These data together with already known sequence data and the arrangement of cfx genes suggested that the Rubisco, fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase and PRK genes are constituents of the same operon. This was further confirmed by trans complementation analyses which indicated that the very similarly organized pHG1-encoded cfx genes additionally present in wild-type strain H16 are functional and also form a common operon. Each operon may also include a glyceraldehyde-3-phosphate dehydrogenase gene. Thus, the duplicated cfx operons of A. eutrophus H16 are large transcriptional units comprising at least about 8 kilobase pairs (kb) and possibly as much as 11 kb.