Isebrand Prinsloo

Selectins and anti-CD15 (Lewis x/a) antibodies transmit activation signals in Hodgkin's lymphoma–derived cell lines

OBJECTIVE The CD15 (Lewis x) cell surface oligosaccharide moiety is expressed in a variety of normal and tumor cells and recognized by selectins. The detection of CD15 on malignant Hodgkin-Reed-Sternberg (HRS) cells serves as a diagnostic marker of Hodgkins lymphoma (HL). Retrospective studies suggest that the expression of nonsialylated CD15 molecules on HRS cells has a positive prognostic value while presence of sialylated CD15 may correlate with a poor outcome. However, the relevance of the CD15 antigen expression to the pathobiology of the disease is not clear. In this work, we studied the contribution of CD15 to cell adhesion and the activation of signaling cascades in two HL-derived cell lines, KMH-2 and L428. METHODS Immobilized anti-CD15 monoclonal antibodies and recombinant E- and P-selectins were used to activate KMH-2 and L428 cells. Immunoblotting, immunoprecipitation, and the electrophoretic mobility shift assay were performed to detect tyrosine phosphorylation of c-Cbl, c-Jun nuclear translocation, and AP-1 DNA binding. RESULTS Treatment of cells with antibodies against the sialylated and nonsialylated forms of CD15, or with immobilized selectins, induced changes in cell morphology. Tyrosine phosphorylation of c-Cbl, together with tyrosine phosphorylation of multiple protein substrates, was also induced. In addition, binding of the CD15 molecules induced nuclear translocation of c-Jun and an increase in AP-1 DNA binding activity. CONCLUSIONS We suggest that CD15 has a dual physiological role, both as an adhesion molecule recognized by selectins and as a regulatory molecule upstream to specific intracellular signaling cascades with implications to the pathogenesis of HL.

Neoangiogenesis in squamous cell carcinoma of the larynx - biological and prognostic associations.

Tumor angiogenesis has been related to tumor growth and an increased probability of metastatic spread. Previous studies have led to conflicting views regarding the prognostic significance of angiogenesis in squamous cell carcinoma of the head and neck. To evaluate the role of tumor angiogenesis in the biology of squamous cell carcinoma of the larynx, we quantified the microvascular network in 59 primary laryngeal carcinomas and looked for an association with outcome. Microvessels were stained immunohistochemically using antibodies for factor VIII-related antigen and the antibody JC70 (CD-31). In each case, microvessels were counted in three fields at x200 magnification, in areas of most intense neovascularization. We found a significantly higher number of microvessels in tumors showing deeper levels of invasion.

Apoptotic Index as a Prognostic Factor in Hodgkin's Disease

Hodgkins disease (HD) is an unusual malignant neoplasm, mainly because of the rarity of tumor cells in the diseased tissues, but also due to a relatively favorable response to treatment. In a previous study, we have shown a variable degree of apoptosis in lymph nodes from HD patients. We now looked for clinicopathological correlations of apoptosis with special emphasis on the prognosis in this disease. A retrospective study of 92 patients was carried out, using in situ end labelling of DNA fragments and an apoptosis detection kit. An apoptotic index (Al) was calculated in each case, as the percentage of apoptotic Hodgkin-Reed-Sternberg cells out of the total number of tumor cells in 10 selected high power fields. An association between a high Al and advanced stages was noted. A Kaplan-Meier analysis showed a negative correlation between Al and survival (p=0.05). In a multivariable analysis adjusting for Ann Arbor stage, a high Al carried a 3.27 fold risk of dying of HD (OR=3.27; Cl=0.89-11.94). However, in our limited cohort of HD patients, Al was not an independent prognostic factor. The results of this study confirm the important role played by apoptosis in HD and suggest that the apoptotic index is probably a negative prognostic marker in this disease. Its assessment in patients with HD may provide a new, important clinical tool.

Differential Expression of Sialyl and Non-Sialyl-CD15 Antigens on Hodgkin-Reed-Sternberg Cells: Significance in Hodgkin's Disease

CD15 expression has been used for years to confirm the diagnosis of Hodgkins disease (HD). Little is, however, known on the relevance of the CD15 antigen to the pathobiology of the disease and there is conflicting evidence as to the prognostic value of its expression. To investigate the significance of the differential expression of CD 15 in Hodgkins disease, a retrospective study of 102 patients with “classical” Hodgkins disease was performed. Immuno-histochemical studies were carried out using antibodies against two types of CD 15: non-sialylated CD15 (LeuM1 and 80H5) and sialylated CD15 (FH6 and CSLEX1). Cases that were negative for non-sialylated CD 15 or positive for the sialylated variant were stained again following neuraminidase pretreatment. The cohort included 27 patients in whom sequential biopsies were available. Both CD15 expression in its non-sialylated form and absence of sialyl-CD15 expression correlate with a favorable outcome. Subsequent biopsies show a preferential expression of sialyl-CD15, notably in bone marrow metastases. Our findings suggest that, in the progression of HD towards a widely disseminated disease, the LewisX moiety of the CD 15 antigen on the tumor cells acquires a sialyl-group. This change may confer on the tumor cells the capacity to metastasize.

Apoptosis (programmed cell death) in colonic cells: from normal to transformed stage

We determined apoptosis in whole rat colonic tissue and in isolated colonocytes from the various rat crypt regions in preneoplastic stages up to frank neoplasia following administration of the procarcinogen, dimethylhydrazine (DMH). Apoptotic cells were determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-method, by evaluating sections stained with hematoxylin and eosin, and caspase-1 immunostaining. Apoptotic cells in whole colonic tissue from untreated rats were confined to the upper crypt while, in DMH-treated rats apoptotic and caspase-1 positive cells were located in the crypt proliferative regions. Numerous apoptotic and caspase-1-positive cells were found in sections from early tumors while in the delayed tumors, apoptotic-positive cells were absent and number of caspase-1-positive cells was negligible. A marked reduction in the apoptotic index along the crypt was observed in isolated transformed colonic cells, this was not the case for caspase-1-positive cells. We conclude that: (i) in colorectal tumors at progressive stage apoptosis is altered, (ii) the mechanistic alteration in apoptosis may be located between caspase-1-protease activity and the fragmentation process of DNA.

Extensive apoptotic death of rat colonic cells deprived of crypt habitat

Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell‐to‐cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD‐CMK) inhibitor of interleukin‐1β‐converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium‐orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic‐labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD‐CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE‐mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype. J. Cell. Physiol. 177:377–386, 1998.

Induction of interleukin-1α production in murine sertoli cells by interleukin-1.

Abstract In the present study we examined the involvement of interleukin (IL)-1α, -1β, FSH, and lipopolysaccharide (LPS) in the regulation of IL-1α and -1β production by Sertoli cells under in vitro conditions. Sertoli cell cultures from immature mice produced constitutively basal levels of intracellular IL-1α. Stimulation of Sertoli cell cultures with LPS (5 μg/ml) resulted in a maximal production of intracellular IL-1α 2 h after the stimulation. Thereafter, these levels decreased but remained significantly higher within 24 h after stimulation than those in control cultures. The effect of LPS on IL-1α production was dose dependent. FSH did not show any effect on intracellular IL-1α production by Sertoli cells. IL-1α could not be detected in supernatants of unstimulated or stimulated Sertoli cell cultures. Sertoli cell cultures stimulated with recombinant IL-1α induced optimal intracellular levels of IL-1α within 2 h of stimulation. These levels remained high 24 h after stimulation. However, stimulation of Sertoli cell cultures with IL-1β induced a peak of IL-1α production 8 h after stimulation. These levels decreased 24 h after the stimulation but were still found to be significantly higher than those in control cultures. The addition of IL-1 receptor antagonist (IL-1ra) to Sertoli cell cultures did not significantly alter their capacity to produce IL-1α. However, the stimulatory effects of recombinant IL-1α on IL-1α production by Sertoli cell cultures were reversed by the concomitant addition of recombinant IL-1ra. No immunoreactive IL-1β could be detected in lysates or conditioned media of immature murine Sertoli cells under any of the stimulatory conditions outlined. Our results may suggest the involvement of physiological (IL-1) and pathophysiological factors (LPS) in the regulation of spermatogenesis and spermiogenesis processes and male fertility.

Is Classical Hodgkin's Disease Indeed a Single Entity?

We present a retrospective clinicopathological study on the significance of the histologic type of classical Hodgkins disease (HD) in a cohort of patients from southern Israel. This was performed to critically evaluate the generally accepted view that classical HD is a single clinicopathological entity and the resultant impression that its segregation into four different histologic types remains essential only for the pathologist in his diagnostic endeavor. We confirmed the existence of a uniform response of nodular sclerosis (NS), and mixed cellularity-lymphocyte depletion (MC-LD)-HD to treatment, consideration being given to other classical prognostic factors. We also accept the fact that histological type is not a significant independent factor in terms of survival. Our findings, however, do suggest that NS-HD, on the one hand, and MC-LD-HD, on the other, are distinct biologic entities. Cases of NS differ significantly from those of MC-LD-HD with regard to sex and age distribution, and in the expression of several antigens and gene products, including sialylated-CD15, CD30, LMP1 and the p53 and mdm-2 gene products.

Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC).

Hodgkins lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkins lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (LewisX), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system.

Lymphangiogenesis in Classical Hodgkin Lymphoma - Preliminary Study with Clinicopathological Correlations.

A role for lymphangiogenesis in metastatic breast and prostate cancers has been suggested recently. The relevance of lymphangiogenesis in cancer as a rule, and more specifically in classical Hodgkin lymphoma, is poorly understood in comparison with that of angiogenesis. In a preliminary (pilot) study we have investigated the role of lymphatic vessels growth in 19 cases of classical Hodgkin lymphoma stained with the D2-40 (podoplanin) antibody. In each case, three lymphatic vessels hot spots were scrutinized twice. Of the 57 hot spots thus identified, we chose 15 at random for photography, microvessel counting and image analysis. We determined the mean perimeter, surface area, major axis length and complexity factor for each hot spot and correlated them with clinical and biological features of classical Hodgkin lymphoma. No correlations were found with clinical features. No associations were noted with the standard immuno-markers of classical Hodgkin lymphoma. However, significant inverse correlations were shown with pRb, BAX and IκB-α expression. The mean lymphatic major axis length was inversely correlated with the complexity factor. Last, we carried out an additional clinicopathological correlation of the expression of pRb, BAX and IκB-α in a cohort of classical Hodgkin lymphoma patients previously published.