Stromal expression of hypoxia inducible factor 2a (HIF-2a) and carbonic anhydrase 9 (CA9) are ass... more Stromal expression of hypoxia inducible factor 2a (HIF-2a) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2a-and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5-8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2a, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2a and CA9 stromal-positive CRCs was associated with wild-type TP53 (P ¼ 0.001 and P ¼ 0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P ¼ 0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis. These results suggest that the biology of CRC cells can be modified by alterations in the tumour microenvironment under conditions of tumour hypoxia.
SummaryCultured human endothelial cells were infected with human cytomegalovirus AD 169 and Kerr.... more SummaryCultured human endothelial cells were infected with human cytomegalovirus AD 169 and Kerr. The infection resulted in the appearance of viral antigens in the nuclei of about 10% of the endothelial cells and in the concomittant disappearance of vWF from the infected cells. No differences were observed between endothelial cells from different sources (umbilical cord veins or arteries, adult veins).
Nuclei, isolated from paraffin‐embedded tissue, were stained with propidium iodide (PI) and found... more Nuclei, isolated from paraffin‐embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA‐derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffinembedded normal and tumor tissue of the same specimen were mixed. With this ...
Recent flow cytometric (FCM) studies have indicated the prognostic value of S-phase cells (SPF) i... more Recent flow cytometric (FCM) studies have indicated the prognostic value of S-phase cells (SPF) in lung cancer. More refined cytokinetic analysis can be obtained by dual-parameter FCM, labeling S-phase cells with 5-bromodeoxyuridine (BrdUrd), which can be detected using a monoclonal antibody (MoAb) to BrdUrd. Tumor cells obtained through bronchoscopic brush were incubated for 1 hr in RPMI 1640 medium with 10% fetal calf serum and 10 microM BrdUrd. After fixation in ethanol, pepsin treatment, and DNA denaturation, the nuclei were stained with anti-BrdUrd MoAb and propidium iodide. From 14 of 20 patients, sufficient material was obtained (three adenocarcinoma and seven squamous cell, one giant cell, and three small cell carcinoma). The measured SPF ranged from 5.2% to 26%. The labeling index (LI), calculated as the ratio of the number of BrdUrd-labeled cells to the total number of aneuploid cells, or diploid cells in the case of a diploid tumor, ranged from 1.2% to 16.7%; LI and SPF c...
Increased systemic levels of endotoxin have been detected in human alcoholics and are thought to ... more Increased systemic levels of endotoxin have been detected in human alcoholics and are thought to be derived from the gut. Although a 'leaky gut' is considered to be a necessary factor for alcohol-induced endotoxemia followed by chronic liver injury, the effects of low concentrations of ethanol on intestinal epithelial cells have not been fully understood. The aim of this study was to evaluate intestinal epithelial cell death induced by acute, low concentrations of ethanol in an in vitro system. The human intestinal Caco-2 cell line was incubated with 0%, 5%, 10% ethanol for up to 3 h. Phosphatidylserine (PS) externalization, caspase-mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were evaluated using flow cytometry. The caspase inhibitor zVAD-fmk was used to test the role of caspases in ethanol-induced cell death. Treatment with 5% and 10% ethanol for 3 h led to a gradual increase in PS externalization. Caspase-mediated CK18 was significantly enhanced as early as 1 h after 10% ethanol incubation, while DNA fragmentation was detected from 2 h onwards. Not only caspase activation but also both PS externalization and DNA fragmentation were completely prevented by pretreatment with the caspase inhibitor. Apoptotic cell death in confluent Caco-2 cells was induced by acute and low concentrations of ethanol. These results suggest that clinically achievable doses of ethanol impair intestinal barrier function by induction of apoptosis in intestinal epithelial cells. This impairment of the barrier function would allow endotoxin to enter the circulation and evoke hepatic inflammation.
Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow... more Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow cytometric DNA content. In order to increase the sensitivity of the assay we tried to enrich for the neoplastic cells by bivariate analysis using a polyclonal anti-nucleolar antibody (AN-AB) and the forward scatter (FSC). DNA aneuploidy was found to be present in 67 of all 137 cases (49%), in 24 cases only demonstrable by dual-parameter analysis. The DNA index varied from 0.69 to 1.89 with a total of 22 hypo-diploid cases. The number of aneuploid nuclei exceeded the expected frequency of Reed-Sternberg (RS) and Hodgkin (H) cells in most of the analysed specimens. In conclusion, flow cytometry in Hodgkin's disease appears to give useful information regarding the ploidy status and evidence has been provided that the malignant cell population in Hodgkin's disease is not limited to the classical RS/H cells.
In Hodgkin's disease DNA aneuploidy is not a prognostic factor. However, the prognostic s... more In Hodgkin's disease DNA aneuploidy is not a prognostic factor. However, the prognostic significance of DNA content in Hodgkin's disease may be missed by either intratumor DNA heterogeneity or DNA analysis of limited samples. For flow cytometry usually one section of 40-60 microns is used for the analysis. In breast cancer this proved to be insufficient. In Hodgkin's disease no data are available. Therefore, we examined if analysis of more sections does increase the yield of aneuploidy. Archival, formalin-fixed, parafin embedded tissues were used. From 13 patients four sections of 50 microns could be analysed for DNA content. In 12 of 13 patients the results were consistent in all four sections of one patient case; seven diploid, four aneuploid and one multiploid. In one case ploidy status changed: two sections were diploid and two were aneuploid. The DNA-index of the aneuploid samples ranged from 0.75 to 1.38 and varied from 0.02 to 0.14 within one case. The S-phase fraction remained constant within all evaluable cases (sd: 0.5-1.5%), except for one (sd: 4.7%). In conclusion, in Hodgkin's disease the ploidy status of the first section can be regarded to represent the whole tissue sample. Therefore, the absence of prognostic value of ploidy status is not explained by sampling errors in tissues analysed.
Immunoreactivity of 1116 NS 19‐9 monoclonal antibody defined monosialoganglioside (gastrointestin... more Immunoreactivity of 1116 NS 19‐9 monoclonal antibody defined monosialoganglioside (gastrointestinal cancer‐associated antigen, GICA) has been studied in a series of colorectal carcinoma patients of a multicentre prospective controlled trial in order to assess its correlation with parameters such as localization, stage histopathological characteristics and DNA flow cytometry. GICA could be detected in 60% of the carcinomas, but no correlation was observed between its status of immunoreactivity and any of the parameters studied. It is concluded that, though study of the expression of the monosialganglioside may be worth while in relation to fundamental aspects of behaviour of colorectal carcinomas, the significance of its immunohistochemical detection in a diagnostic or prognostic sense is limited.
Immunohistochemistry of the S phase related proliferating cell nuclear antigen (PCNA) was studied... more Immunohistochemistry of the S phase related proliferating cell nuclear antigen (PCNA) was studied as an alternative to ex-vivo bromodeoxyuridine (BrdU) immunohistochemistry for assessment of human colonic cell proliferation. From 16 subjects without colonic disease biopsy specimens were collected from five different sites along the colorectum and processed for BrdU and PCNA immunohistochemistry. The mean proliferation index of
We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of... more We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of DNA flow cytometry. We report the correlations between DNA index (DNI) and fraction of cells in S-phase (SPF) and other prognostic parameters. Both univariately and in a Cox model, the predictive power of these factors is evaluated after a follow-up of more than 10 years. There are strong correlations between DNI and SPF (P = 0.0001) and between flow cytometry parameters and clinical and histopathological factors such as axillary lymph node involvement, tumour size and histological grade. In univariate analysis DNI fails to provide prognostic information, whereas SPF turns out to be able to differentiate between patients at high and low risk for relapse and death (P = 0.002). In the multivariate Cox model, too, SPF is an important prognostic parameter with respect to patient survival (relative risk: +86%), only surpassed by nodal involvement. DNI, however, turns out to be an independent predictor of relapse free survival and distant recurrence free survival. By combination of DNI and SPF, patients can be divided into three prognostic subgroups. We conclude that data from DNA flow cytometry can be of great importance for the decision on the level of aggressiveness of adjuvant therapy for an individual patient and therefore may help to avoid overtreatment and toxicity.
In the mouse mutant curly tail, the phenotypes spina bifida and curled tail result from a delay i... more In the mouse mutant curly tail, the phenotypes spina bifida and curled tail result from a delay in closure of the posterior neuropore (PNP). At the developmental stage when this delay can first be recognized, the caudal region of the embryo demonstrates a transiently enhanced curvature of the body axis which likely inhibits elevation, convergence, and fusion of the neural folds. The enhanced curvature is thought to be the result of a decreased proliferation in the ventrally located gut endoderm and notochord, together with a normal proliferation of the overlying neuroepithelium of the PNP. However, the proliferation defect and the enhanced curvature were originally demonstrated at the same developmental stage, while it is expected that reduced proliferation should precede enhanced curvature and delayed PNP closure. The caudal region originates from the tail bud and we therefore propose that the enhanced curvature is induced by a disturbed dorso-ventral proliferation pattern in the tail bud. Using flow cytometry, proliferation patterns were determined separately for the dorsal and ventral halves of the tail bud of curly tail and of control embryos as well as of recombinant embryos having the curly tail phenotype with a genetic background which is matched to the BALB/c control strain. In general, it appeared that about half of the cell cycle duration in tail bud cells was occupied by S phase, about 40% by G0/G1 and the rest by G2/M. For the control embryos, no dorso-ventral differences in relative phase duration were demonstrated. However, curly tail and recombinant embryos at the 21-25 somite stage, prior to the onset of enhanced curvature, exhibited ventrally a higher proportion of G0/G1 phase cells than dorsally, and a complementary relationship for S phase cells. We interpret these observations as indicating a prolonged G1 phase at the ventral side of the tail bud, resulting in a prolongation of the cell cycle and thus a decreased proliferation. In 26-30 somite stage embryos, prior to the normalization of curvature in curly tail embryos, the dorso-ventral proliferation balance was re-established. We conclude that a reduced proliferation in the ventral part of the tail bud of the curly tail embryo precedes both the onset of enhanced curvature and the previously observed reduction in proliferation of the hindgut and notochord, and is a likely candidate for an early event in the pathogenetic sequence leading to the curly tail phenotype.
In order to determine the effect of oral administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA;... more In order to determine the effect of oral administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA; dose-level: 1.5% BHA of the diet) on arachidonic acid (AA) and linoleic acid (LA) metabolism in correlation with changes in gastrointestinal cell kinetics, we coadministered two inhibitors of prostaglandin H synthase, acetylsalicylic acid (ASA) and indomethacin (IM), to rats. Coadministration of ASA (0.2%) and IM (0.002%) in the drinking water, resulted in a significant reduction of the BHA-induced enhancement of cell proliferation in forestomach and glandular stomach. ASA completely counteracted the effect of BHA on labeling indices in colon/rectum whereas IM exhibited no effect in this organ. Both inhibitors had no direct effect on cell kinetics in the control groups. ASA, and to a lesser degree IM, inhibited prostaglandin E2 release in all tissues examined. Whereas ASA did inhibit lipoxygenase-mediated metabolism of AA in forestomach tissue, ASA did not affect the release of AA- and LA-derived hydroxy fatty acids in glandular stomach and colon/rectum. IM did not affect lipoxygenase production. BHA, however, appeared to be a strong inhibitor of both routes of AA metabolism. While ASA nor IM affected LA metabolism, BHA inhibited both prostaglandin H synthase-mediated and lipoxygenase-mediated metabolism of AA and LA. A causal role of AA or LA metabolites in the process of cell proliferation enhancement induced by BHA, can therefore be excluded. Prostaglandin H synthase may, however, be involved in BHA activation by converting the hydroquinone metabolite of BHA to the corresponding quinone by redox cycling, which is probably accompanied by reactive intermediate production.
A multivariate analysis of the pathologic data of 350 patients with primary colorectal cancer was... more A multivariate analysis of the pathologic data of 350 patients with primary colorectal cancer was performed. In addition to conventional parameters such as shape and size of the primary tumor, central node involvement, angioinvasive growth, grade, and stage, new variables such as the immunoreactivity patterns of carcinoembryonic antigen (CEA), CA 19-9, mucin, serotonin, secretory component (SC), and the DNA index were tested for their potential prognostic value. Every variable except CA 19-9, serotonin, and DNA showed significant prognostic information in univariate analysis. However, in the multivariate analysis stage was the predictive factor with the highest hazard ratio, but absence of central node involvement, tumors with diameters between 3.5 cm and 6 cm, exophytic tumor growth, well-differentiated tumors, tumors with CEA immunoreactivity, absence for staining with serotonin, and diploid tumors also were included in the relative risk model. Thus, the aforementioned variables appear to play a role in the establishment of a prognostic index.
Summary— Olomoucine (2‐(2‐hydroxyethylamino)‐6‐benzylamino‐9‐methylpurine) has been recently desc... more Summary— Olomoucine (2‐(2‐hydroxyethylamino)‐6‐benzylamino‐9‐methylpurine) has been recently described as a competitive inhibitor (ATP‐binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5 p35 kinase and the ERK1AP‐kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin‐i...
In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcino... more In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow
Percentage of proliferating choanocytes in Halisarca caerulea specimens, incubated with BrdU for ... more Percentage of proliferating choanocytes in Halisarca caerulea specimens, incubated with BrdU for either 6 hours or 10 hours
Stromal expression of hypoxia inducible factor 2a (HIF-2a) and carbonic anhydrase 9 (CA9) are ass... more Stromal expression of hypoxia inducible factor 2a (HIF-2a) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2a-and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5-8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2a, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2a and CA9 stromal-positive CRCs was associated with wild-type TP53 (P ¼ 0.001 and P ¼ 0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P ¼ 0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis. These results suggest that the biology of CRC cells can be modified by alterations in the tumour microenvironment under conditions of tumour hypoxia.
SummaryCultured human endothelial cells were infected with human cytomegalovirus AD 169 and Kerr.... more SummaryCultured human endothelial cells were infected with human cytomegalovirus AD 169 and Kerr. The infection resulted in the appearance of viral antigens in the nuclei of about 10% of the endothelial cells and in the concomittant disappearance of vWF from the infected cells. No differences were observed between endothelial cells from different sources (umbilical cord veins or arteries, adult veins).
Nuclei, isolated from paraffin‐embedded tissue, were stained with propidium iodide (PI) and found... more Nuclei, isolated from paraffin‐embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA‐derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffinembedded normal and tumor tissue of the same specimen were mixed. With this ...
Recent flow cytometric (FCM) studies have indicated the prognostic value of S-phase cells (SPF) i... more Recent flow cytometric (FCM) studies have indicated the prognostic value of S-phase cells (SPF) in lung cancer. More refined cytokinetic analysis can be obtained by dual-parameter FCM, labeling S-phase cells with 5-bromodeoxyuridine (BrdUrd), which can be detected using a monoclonal antibody (MoAb) to BrdUrd. Tumor cells obtained through bronchoscopic brush were incubated for 1 hr in RPMI 1640 medium with 10% fetal calf serum and 10 microM BrdUrd. After fixation in ethanol, pepsin treatment, and DNA denaturation, the nuclei were stained with anti-BrdUrd MoAb and propidium iodide. From 14 of 20 patients, sufficient material was obtained (three adenocarcinoma and seven squamous cell, one giant cell, and three small cell carcinoma). The measured SPF ranged from 5.2% to 26%. The labeling index (LI), calculated as the ratio of the number of BrdUrd-labeled cells to the total number of aneuploid cells, or diploid cells in the case of a diploid tumor, ranged from 1.2% to 16.7%; LI and SPF c...
Increased systemic levels of endotoxin have been detected in human alcoholics and are thought to ... more Increased systemic levels of endotoxin have been detected in human alcoholics and are thought to be derived from the gut. Although a 'leaky gut' is considered to be a necessary factor for alcohol-induced endotoxemia followed by chronic liver injury, the effects of low concentrations of ethanol on intestinal epithelial cells have not been fully understood. The aim of this study was to evaluate intestinal epithelial cell death induced by acute, low concentrations of ethanol in an in vitro system. The human intestinal Caco-2 cell line was incubated with 0%, 5%, 10% ethanol for up to 3 h. Phosphatidylserine (PS) externalization, caspase-mediated cytokeratin 18 (CK18) cleavage, and DNA fragmentation were evaluated using flow cytometry. The caspase inhibitor zVAD-fmk was used to test the role of caspases in ethanol-induced cell death. Treatment with 5% and 10% ethanol for 3 h led to a gradual increase in PS externalization. Caspase-mediated CK18 was significantly enhanced as early as 1 h after 10% ethanol incubation, while DNA fragmentation was detected from 2 h onwards. Not only caspase activation but also both PS externalization and DNA fragmentation were completely prevented by pretreatment with the caspase inhibitor. Apoptotic cell death in confluent Caco-2 cells was induced by acute and low concentrations of ethanol. These results suggest that clinically achievable doses of ethanol impair intestinal barrier function by induction of apoptosis in intestinal epithelial cells. This impairment of the barrier function would allow endotoxin to enter the circulation and evoke hepatic inflammation.
Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow... more Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow cytometric DNA content. In order to increase the sensitivity of the assay we tried to enrich for the neoplastic cells by bivariate analysis using a polyclonal anti-nucleolar antibody (AN-AB) and the forward scatter (FSC). DNA aneuploidy was found to be present in 67 of all 137 cases (49%), in 24 cases only demonstrable by dual-parameter analysis. The DNA index varied from 0.69 to 1.89 with a total of 22 hypo-diploid cases. The number of aneuploid nuclei exceeded the expected frequency of Reed-Sternberg (RS) and Hodgkin (H) cells in most of the analysed specimens. In conclusion, flow cytometry in Hodgkin's disease appears to give useful information regarding the ploidy status and evidence has been provided that the malignant cell population in Hodgkin's disease is not limited to the classical RS/H cells.
In Hodgkin's disease DNA aneuploidy is not a prognostic factor. However, the prognostic s... more In Hodgkin's disease DNA aneuploidy is not a prognostic factor. However, the prognostic significance of DNA content in Hodgkin's disease may be missed by either intratumor DNA heterogeneity or DNA analysis of limited samples. For flow cytometry usually one section of 40-60 microns is used for the analysis. In breast cancer this proved to be insufficient. In Hodgkin's disease no data are available. Therefore, we examined if analysis of more sections does increase the yield of aneuploidy. Archival, formalin-fixed, parafin embedded tissues were used. From 13 patients four sections of 50 microns could be analysed for DNA content. In 12 of 13 patients the results were consistent in all four sections of one patient case; seven diploid, four aneuploid and one multiploid. In one case ploidy status changed: two sections were diploid and two were aneuploid. The DNA-index of the aneuploid samples ranged from 0.75 to 1.38 and varied from 0.02 to 0.14 within one case. The S-phase fraction remained constant within all evaluable cases (sd: 0.5-1.5%), except for one (sd: 4.7%). In conclusion, in Hodgkin's disease the ploidy status of the first section can be regarded to represent the whole tissue sample. Therefore, the absence of prognostic value of ploidy status is not explained by sampling errors in tissues analysed.
Immunoreactivity of 1116 NS 19‐9 monoclonal antibody defined monosialoganglioside (gastrointestin... more Immunoreactivity of 1116 NS 19‐9 monoclonal antibody defined monosialoganglioside (gastrointestinal cancer‐associated antigen, GICA) has been studied in a series of colorectal carcinoma patients of a multicentre prospective controlled trial in order to assess its correlation with parameters such as localization, stage histopathological characteristics and DNA flow cytometry. GICA could be detected in 60% of the carcinomas, but no correlation was observed between its status of immunoreactivity and any of the parameters studied. It is concluded that, though study of the expression of the monosialganglioside may be worth while in relation to fundamental aspects of behaviour of colorectal carcinomas, the significance of its immunohistochemical detection in a diagnostic or prognostic sense is limited.
Immunohistochemistry of the S phase related proliferating cell nuclear antigen (PCNA) was studied... more Immunohistochemistry of the S phase related proliferating cell nuclear antigen (PCNA) was studied as an alternative to ex-vivo bromodeoxyuridine (BrdU) immunohistochemistry for assessment of human colonic cell proliferation. From 16 subjects without colonic disease biopsy specimens were collected from five different sites along the colorectum and processed for BrdU and PCNA immunohistochemistry. The mean proliferation index of
We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of... more We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of DNA flow cytometry. We report the correlations between DNA index (DNI) and fraction of cells in S-phase (SPF) and other prognostic parameters. Both univariately and in a Cox model, the predictive power of these factors is evaluated after a follow-up of more than 10 years. There are strong correlations between DNI and SPF (P = 0.0001) and between flow cytometry parameters and clinical and histopathological factors such as axillary lymph node involvement, tumour size and histological grade. In univariate analysis DNI fails to provide prognostic information, whereas SPF turns out to be able to differentiate between patients at high and low risk for relapse and death (P = 0.002). In the multivariate Cox model, too, SPF is an important prognostic parameter with respect to patient survival (relative risk: +86%), only surpassed by nodal involvement. DNI, however, turns out to be an independent predictor of relapse free survival and distant recurrence free survival. By combination of DNI and SPF, patients can be divided into three prognostic subgroups. We conclude that data from DNA flow cytometry can be of great importance for the decision on the level of aggressiveness of adjuvant therapy for an individual patient and therefore may help to avoid overtreatment and toxicity.
In the mouse mutant curly tail, the phenotypes spina bifida and curled tail result from a delay i... more In the mouse mutant curly tail, the phenotypes spina bifida and curled tail result from a delay in closure of the posterior neuropore (PNP). At the developmental stage when this delay can first be recognized, the caudal region of the embryo demonstrates a transiently enhanced curvature of the body axis which likely inhibits elevation, convergence, and fusion of the neural folds. The enhanced curvature is thought to be the result of a decreased proliferation in the ventrally located gut endoderm and notochord, together with a normal proliferation of the overlying neuroepithelium of the PNP. However, the proliferation defect and the enhanced curvature were originally demonstrated at the same developmental stage, while it is expected that reduced proliferation should precede enhanced curvature and delayed PNP closure. The caudal region originates from the tail bud and we therefore propose that the enhanced curvature is induced by a disturbed dorso-ventral proliferation pattern in the tail bud. Using flow cytometry, proliferation patterns were determined separately for the dorsal and ventral halves of the tail bud of curly tail and of control embryos as well as of recombinant embryos having the curly tail phenotype with a genetic background which is matched to the BALB/c control strain. In general, it appeared that about half of the cell cycle duration in tail bud cells was occupied by S phase, about 40% by G0/G1 and the rest by G2/M. For the control embryos, no dorso-ventral differences in relative phase duration were demonstrated. However, curly tail and recombinant embryos at the 21-25 somite stage, prior to the onset of enhanced curvature, exhibited ventrally a higher proportion of G0/G1 phase cells than dorsally, and a complementary relationship for S phase cells. We interpret these observations as indicating a prolonged G1 phase at the ventral side of the tail bud, resulting in a prolongation of the cell cycle and thus a decreased proliferation. In 26-30 somite stage embryos, prior to the normalization of curvature in curly tail embryos, the dorso-ventral proliferation balance was re-established. We conclude that a reduced proliferation in the ventral part of the tail bud of the curly tail embryo precedes both the onset of enhanced curvature and the previously observed reduction in proliferation of the hindgut and notochord, and is a likely candidate for an early event in the pathogenetic sequence leading to the curly tail phenotype.
In order to determine the effect of oral administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA;... more In order to determine the effect of oral administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA; dose-level: 1.5% BHA of the diet) on arachidonic acid (AA) and linoleic acid (LA) metabolism in correlation with changes in gastrointestinal cell kinetics, we coadministered two inhibitors of prostaglandin H synthase, acetylsalicylic acid (ASA) and indomethacin (IM), to rats. Coadministration of ASA (0.2%) and IM (0.002%) in the drinking water, resulted in a significant reduction of the BHA-induced enhancement of cell proliferation in forestomach and glandular stomach. ASA completely counteracted the effect of BHA on labeling indices in colon/rectum whereas IM exhibited no effect in this organ. Both inhibitors had no direct effect on cell kinetics in the control groups. ASA, and to a lesser degree IM, inhibited prostaglandin E2 release in all tissues examined. Whereas ASA did inhibit lipoxygenase-mediated metabolism of AA in forestomach tissue, ASA did not affect the release of AA- and LA-derived hydroxy fatty acids in glandular stomach and colon/rectum. IM did not affect lipoxygenase production. BHA, however, appeared to be a strong inhibitor of both routes of AA metabolism. While ASA nor IM affected LA metabolism, BHA inhibited both prostaglandin H synthase-mediated and lipoxygenase-mediated metabolism of AA and LA. A causal role of AA or LA metabolites in the process of cell proliferation enhancement induced by BHA, can therefore be excluded. Prostaglandin H synthase may, however, be involved in BHA activation by converting the hydroquinone metabolite of BHA to the corresponding quinone by redox cycling, which is probably accompanied by reactive intermediate production.
A multivariate analysis of the pathologic data of 350 patients with primary colorectal cancer was... more A multivariate analysis of the pathologic data of 350 patients with primary colorectal cancer was performed. In addition to conventional parameters such as shape and size of the primary tumor, central node involvement, angioinvasive growth, grade, and stage, new variables such as the immunoreactivity patterns of carcinoembryonic antigen (CEA), CA 19-9, mucin, serotonin, secretory component (SC), and the DNA index were tested for their potential prognostic value. Every variable except CA 19-9, serotonin, and DNA showed significant prognostic information in univariate analysis. However, in the multivariate analysis stage was the predictive factor with the highest hazard ratio, but absence of central node involvement, tumors with diameters between 3.5 cm and 6 cm, exophytic tumor growth, well-differentiated tumors, tumors with CEA immunoreactivity, absence for staining with serotonin, and diploid tumors also were included in the relative risk model. Thus, the aforementioned variables appear to play a role in the establishment of a prognostic index.
Summary— Olomoucine (2‐(2‐hydroxyethylamino)‐6‐benzylamino‐9‐methylpurine) has been recently desc... more Summary— Olomoucine (2‐(2‐hydroxyethylamino)‐6‐benzylamino‐9‐methylpurine) has been recently described as a competitive inhibitor (ATP‐binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5 p35 kinase and the ERK1AP‐kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin‐i...
In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcino... more In a series of 44 bronchial biopsies from patients suspected of having endobronchial lung carcinoma, the validity of proliferating cell nuclear antigen (PCNA) and Ki67 antigen as proliferative indicators was evaluated in ethanol fixed, paraffin embedded tissue. The percentages of cells positive for these markers were compared to the in vivo bromodeoxyuridine (BrdU) labelling index. A good correlation was found between PCNA immunoreactivity and BrdU labelling index, while Ki67antigen expression showed a significant relation with BrdU labelling index and with PCNA expression. All three parameters showed a trend towards similar values for the individual cases. Based on the fact that Ki67 antigen is expressed in all cycling cells, whereas replicon-associated PCNA and BrdU only reflect the S-phase fraction, the differences between Ki67-antigen scores on the one hand and BrdU and PCNA scores on the other were smaller than expected. In order to determine the degree of concordance between immunohistochemically and flow cytometrically detected proliferation variables, BrdU incorporation was measured using both methods in duplicate bronchial specimens. Discrepancies in labelling indices were observed predominantly in DNA diploid samples, with consistently lower values in the flow
Percentage of proliferating choanocytes in Halisarca caerulea specimens, incubated with BrdU for ... more Percentage of proliferating choanocytes in Halisarca caerulea specimens, incubated with BrdU for either 6 hours or 10 hours
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