Jörn Sträter

CD95 (APO-1/Fas)-mediated apoptosis in colon epithelial cells: A possible role in ulcerative colitis

BACKGROUND & AIMS Ligation of CD95 (APO-1/Fas) by antibody or CD95 ligand (CD95L) induces apoptosis and, in some cell lines, growth. Normal colonic epithelial cells constitutively express CD95. The function of CD95 on colonocytes is unknown. The aim of this study was to elucidate the role of epithelial CD95 in the normal colon and in ulcerative colitis. METHODS Intact colonic crypts were isolated, and the effects of CD95 ligation in vitro were studied. CD95L-expressing cells and apoptotic cells were detected in situ by RNA hybridization, immunohistochemistry, and DNA nick end labeling. RESULTS On CD95 ligation, isolated colonic crypt cells underwent apoptosis within 4 hours. No growth-promoting effect was observed. In normal colon, CD95L expression was restricted to few mononuclear cells randomly scattered within the lamina propria. Therefore, the CD95-CD95L system is very unlikely to operate in the regeneration of the colonic epithelium. However, in ulcerative colitis, the number of interstitial CD95L+ cells and the frequency of apoptosis in both lamina propria and epithelium were increased considerably. Further, a focal association of subepithelial CD95L+ mononuclear cells and epithelial apoptosis was observed. CONCLUSIONS In ulcerative colitis, soluble CD95L-mediated epithelial apoptosis may lead to a breakdown of the epithelial barrier function facilitating the invasion of pathogenic microorganisms.

In situ detection of enterocytic apoptosis in normal colonic mucosa and in familial adenomatous polyposis.

Physiological regeneration of colonic epithelium entails proliferation at the crypt base and cell loss by shedding or cell death. The aim of this study was to localise and assess the rate of apoptosis in normal and neoplastic colonic epithelium with respect to zones of proliferation. Familial adenomatous polyposis (FAP) was chosen as a model to study neoplastic transformation of colonic mucosa at an early stage. Apoptotic cells were detected in situ by TdT-mediated biotin-dUTP nick end labelling (TUNEL) in parallel to cells in cycle determined by Ki-67 immunohistochemistry using the monoclonal antibody MiB-1. By detection of genomic fragmentation, two different patterns of enterocytic apoptosis emerged: (a) apoptotic bodies being engulfed by adjacent epithelial cells, and (b) apoptotic cells with only subtle morphological changes being extruded into the gut lumen. The engulfment pattern was seen predominantly in the crypts of the normal colonic mucosa and, although very rare, was clearly confined to the basal proliferation compartment. The extrusion pattern was restricted to the luminal mucosal surface. Adenomas of FAP showed highly increased numbers of apoptotic bodies, which were scattered throughout the transformed mucosa. Both patterns of apoptosis were topographically intermingled although the extrusion pattern prevailed at the luminal adenoma surfaces. Whereas cells in cycle were somewhat more numerous in the upper parts of the crypts, apoptosis occurred with increased frequency at sites beneath the proliferation maximum suggesting an inverted direction of epithelial cell migration in adenomas. These results suggest two distinct routes towards enterocytic apoptosis in the colonic mucosa leading to engulfment or extrusion of dying cells. Adenomatous transformation of colon epithelium is associated with a considerable increase of the cellular turnover rate and with a severe disturbance of the microtopographical localisation of birth and death of enterocytes.

Impaired CD95 expression predisposes for recurrence in curatively resected colon carcinoma: clinical evidence for immunoselection and CD95L mediated control of minimal residual disease

Background: Loss of CD95 expression in tumour cells occurs frequently in colon carcinoma and may be associated with disease progression. On the other hand, neo-expression of CD95L in tumour cells may contribute to immune evasion. Aims: We aimed at further exploring the functional role and prognostic significance of the CD95/CD95L death inducing system in colon carcinomas. Patients and methods: CD95 and CD95L expression was examined by immunohistochemistry in 128 R0 resected UICC (International Union against Cancer) stage II/III colon carcinomas and correlated with disease free survival. Results: CD95 expression in tumour cells was observed in only 30 carcinomas (23.4%) whereas the others had at least a minor subpopulation of CD95 negative cells. Loss of CD95 in tumour cells was related to adverse prognosis in uni- and multivariate analysis (p = 0.046 and p = 0.036, respectively). Tumour infiltrating lymphocytes (TIL) were the major source of CD95L in colon carcinomas. CD95L+TIL were present in 83% of cases whereas CD95L was found in tumour cells in only 12% of cases. Moreover, a high rate of CD95L+TIL correlated with prolonged disease free survival in patients with UICC stage II (p = 0.05) but not in those with stage III. Conclusions: Loss of CD95 in tumour cells may be an independent prognostic factor in colon carcinomas. The CD95L counterattack is not a relevant feature in colon carcinoma but CD95L+TIL may contribute to tumour control in the early stages of the disease, exerting a concurrent selection pressure in the direction of CD95 abrogation/resistance.

Induction of Apoptosis Following Blunt Chest Trauma

The cause for the high morbidity of blunt chest trauma is not fully understood. It is still unclear if and to what extent a second insult, e.g., apoptotic tissue damage initiated by the primary insult itself, may contribute to the development of serious complications. This study was done to elucidate whether a pulmonary contusion may induce programmed cell death. Sixty-four Wistar rats were evenly randomized to eight experimental groups: four sets were subjected to a standardized blast wave injury and sacrificed 6, 24, 48, and 72 h after the trauma; four groups served as controls for the same time points. Lung and liver samples were stained (H&E; TUNEL), and PMN infiltration was determined by myeloperoxidase (MPO) activity. Caspase 8 was analyzed by Western blot, and TNF-&agr; plasma levels by ELISA. Postmortem examination revealed bilateral pulmonary contusion in trauma animals with higher (P < 0.05) numbers of apoptotic cells in lung but not in liver tissue as early as 6 h after the injury. This amount gradually increased and reached a maximum after 48 h: 6.8 ± 1.1 apoptotic cells/hpf vs. 0.6 ± 0.06 in controls. Chest trauma caused an increased expression of active caspase 8 in lung but not in liver tissue at 48 and 72 h. TNF-&agr; plasma levels were not different. MPO activity in lung tissue of trauma animals increased (P < 0.05) after 6 h and peaked at 72 h. This study has provided the first evidence that apoptotic cell death in lung tissue is initiated following (experimental) pulmonary contusion. The exact mechanism remains, however, unclear and has to be elucidated further.

CD95 Ligand (CD95L) in Normal Human Lymphoid Tissues: A Subset of Plasma Cells Are Prominent Producers of CD95L

CD95(Fas/APO-1)-ligand (CD95L) mediates apoptosis by trimerization of the CD95 receptor on the surface of sensitive cells. In vitro studies have shown CD95L expression mainly by activated T cells and suggested a role for CD95L in the regulation of immune responses. Little is known, however, about the cellular distribution of CD95L in situ in the normal human immune system. We investigated CD95L expression in tissue sections of the thymus, lymph node, spleen, tonsil, and gastrointestinal tract using in situ hybridization and two monoclonal antibodies. In all these organs, cells expressing CD95L message and protein were scarce and comprised scattered lymphocytes, rare nonlymphoid cells, and a subset of epithelioid endothelial cells. Surprisingly, a subset of plasma cells turned out to be the most prominent producers of CD95L, matching the reports on CD95L in myeloma cells. CD95L+ plasma cells were most numerous in the mucosa-associated lymphoid tissue. This also applied to acquired mucosa-associated lymphoid tissue in chronic gastritis in which CD95L+ plasma cells were found scattered in the lamina propria. Our data suggest that plasma cells as yet may be neglected modulators of immune responses.

MedB-1, a human tumor cell line derived from a primary mediastinal large B-cell lymphoma

Primary mediastinal B‐cell lymphoma is a locally highly aggressive but poorly disseminating tumor composed of medium sized or large cells most probably of thymic medullary origin. It has a mature B‐cell phenotype, typically lacks immunoglobulin expression and has variable defects in expression of HLA‐molecules. We present here a cell line, MedB‐1, derived from such a tumor. As is frequently found in mediastinal B‐cell lymphomas in situ, MedB‐1 is CD10−, CD19+, CD21−, CD22+, CD23+, CD25−, CD37+, CD38−, CD39+, CD40+, CD54+, CD95+. Like the parental tumor, MedB‐1 lacks HLA‐A,B,C α‐chains and β2microglobulin and expresses HLA‐D molecules at decreased levels. Both parental tumor and MedB‐1 cells are clonally related as shown by immunoglobulin heavy chain gene rearrangement analysis. Unlike the parental tumor tissue, the MedB‐1 cell line cytoplasmically expresses IgG/κ in a very small subset of cells under standard culture conditions. MedB‐1 does not contain any Epstein‐Barr virus DNA. In a tissue adhesion assay MedB‐1 cells showed an extensive binding to the medullary region of normal thymus. Altogether, MedB‐1 is a suitable tool for functional and molecular analysis of this distinct lymphoma entity.

Expression and prognostic significance of APAF‐1, caspase‐8 and caspase‐9 in stage II/III colon carcinoma: Caspase‐8 and caspase‐9 is associated with poor prognosis

Apoptosis protease activating factor‐1 (APAF‐1), caspase‐8 and caspase‐9 are important factors in the execution of death signals. To study their prognostic influence in colon carcinoma, expression of APAF‐1, caspase‐8 and caspase‐9 was determined by immunohistochemistry in normal colon mucosa (n = 8) and R0‐resected stage II/III colon carcinomas (n ≥ 124) using a semiquantitative score. Staining results were correlated with disease‐free survival by Kaplan–Meier estimates, and multivariate Cox analyses were performed. In normal colon, APAF‐1 and caspase‐8 are most strongly expressed in the luminal surface epithelium, whereas caspase‐9 is expressed all along the crypt axis. In colon carcinomas, there is considerable variability in the expression of these proapoptotic factors, although complete loss of caspase‐8 and caspase‐9 is rare. APAF‐1 expression did not correlate with disease‐free survival. Instead, both expression of caspase‐9 and high‐level expression of caspase‐8 in a majority of tumor cells were significantly associated with adverse prognosis (p = 0.004 and p = 0.029, respectively). The influence of caspase‐8 expression was mainly seen in patients with stage III colon carcinoma (p = 0.011), whereas the prognostic influence of caspase‐9 expression was significant in stage II cases (p = 0.037) and just failed to be significant in stage III tumors (p = 0.0581). After adjusting for confounding factors in a multivariate Cox analysis, the effect of caspase‐9 in predicting disease‐free survival was confirmed (p = 0.003). Our data suggest that, in colon carcinomas, expression of caspase‐8 and caspase‐9 is significantly associated with poor survival. Caspase‐9 may be an independent prognosticator in colon carcinoma.

Association of time to recurrence with thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression in stage II and III colorectal cancer.

Patients with International Union Against Cancer (UICC) stage IIb and III colon cancer and stage II and III rectal cancer may receive adjuvant chemotherapy with 5-fluorouracil (5-FU). High levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) have been associated with resistance to 5-FU in advanced colorectal cancer. The aim of this study was to investigate the association of TS and DPD mRNA levels with recurrence-free survival in patients with colorectal cancer who are receiving adjuvant 5-FU-based chemotherapy. TS and DPD mRNA quantitation was retrospectively performed in primary colorectal cancer specimens from patients receiving adjuvant 5-FU using a reverse transcription-polymerase chain reaction technique. The median TS mRNA level in patients with a recurrence (n = 142) was 0.68, and in patients without a recurrence (n = 206) the median level was 0.80 (P < 0.01). Patients with a recurrence who had a low TS level (TS ≤ 0.9; n = 102) had a median recurrence-free survival of 18 months (range 3.0 to 54 months), and those with a high TS level (TS > 0.9; n = 40) had a median recurrence-free survival of 11 months (range 1.7 to 53 months; P = 0.0024). There was no difference in the median recurrence-free survival of patients with low and high DPD mRNA levels. The TS mRNA level may be a useful marker to predict the time to recurrence in patients with colorectal cancer who are receiving adjuvant 5-FU treatment.

In chronic pancreatitis, widespread emergence of TRAIL receptors in epithelia coincides with neoexpression of TRAIL by pancreatic stellate cells of early fibrotic areas

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis by cross-linking of the two TRAIL receptors that contain a death domain, TRAIL-R1 and TRAIL-R2. TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. Our aim was to determine the expression of TRAIL and its receptors in normal pancreas and chronic pancreatitis. We applied real-time PCR, immunohisto(cyto)chemistry, and nick-end labeling of apoptoses. In normal pancreas, a minor subset of acinar cells coexpressed TRAIL-R2 and TRAIL-R4, whereas ductular epithelium and interstitial fibroblast-like cells (FLC) expressed TRAIL-R4. TRAIL-R1 and TRAIL-R3 were not detected in normal pancreas. In chronic pancreatitis, the exocrine epithelium strongly expressed TRAIL-R1, -R2, -R4, and, to a lesser extent, TRAIL-R3. Islets focally neoexpressed TRAIL-R1 and -R2 and intensely expressed TRAIL-R4. Changes in TRAIL receptor expression were most pronounced in areas of inflammatory infiltration and active fibrosis. In normal pancreas, expression of TRAIL was low on the mRNA level and undetectable on the protein level. In chronic pancreatitis, FLC in areas of active fibrosis expressed TRAIL. In addition, apoptoses were most numerous in these areas. We show that these FLC are pancreatic stellate cells. Pancreatic stellate cells express TRAIL in vivo and in vitro, and TRAIL expression is enhanced by IFN-γ. Our findings indicate that the TRAIL/TRAIL receptor system is likely to be involved in chronic pancreatitis and suggest that pancreatic stellate cells may directly contribute to acinar regression by inducing apoptosis of parenchymal cells in a TRAIL-dependent manner.

Matrix-comparative genomic hybridization from multicenter formalin-fixed paraffin-embedded colorectal cancer tissue blocks

BackgroundThe identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses.MethodsTo verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III) from systemically advanced (UICC stage IV) colorectal tumours.ResultsThe majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes.ConclusionArchival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.