Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: long-term survival of allogeneic bone marrow and skin grafts.

BALB/c mice were treated with fractionated high dose (3,400 rads) total lymphoid irradiation (TLI), and given semiallogeneic (BALB/c x C57BL/Ka) or allogeneic (C57BL/Ka) bone marrow and/or skin allografts. TLI alone prolonged the mean survival time (m.s.t.) of C57BL/Ka skin grafts to 49.1 days (control, 10.7 days). Shielding of the thymus during TLI produced only a slight increase in graft survival (m.s.t., 19 days). TLI combined with splenectomy was no more effective than TLI alone. Infusion of 10(7) semiallogeneic or allogeneic bone marrow cells after TLI produced stable chimeras in 7/8 and 8/15 recipients, respectively. Chimeras were specifically tolerant to donor tissues, since C57BL/Ka skin grafts were accepted for more than 250 days, but third-party (C3H/He) skin grafts were rejected rapidly. In addition, chimeric lymphocytes responded to C3H/He and C3H. Q but not to C57BL/Ka cells in the one-way mixed leukocyte reactions. BALB/c C57BL/Ka chimeras showed no clinical evidence of graft vs. host disease. These findings may have application of clinical organ transplantation, since (a) the recipient treatment (TLI) has already been shown to be safe in humans, (b) donors and recipients can be completely allogeneic, and (c) bone marrow and skin graft survival was permanent (greater than 250 days).

Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation.

Bone marrow (BM) and skin allografts from C57BL/Ka (H-2b/b) mice were transplanted to BALB/c (H-2d/d) recipients treated with total lymphoid irradiation (TLI), whole-body irradiation (WBI), or fractionated thymic irradiation TLI prolonged skin allograft survival about five times as long as that in untreated controls, and allowed for permanent engraftment of BM cells in approximately equal to 90% of recipients. None of the BM recipients showed clinical signs of graft-versus-host disease (GVHD) (diarrhea, weight loss, hunched back, etc.). On the other hand, recipients given WBI and allogeneic BM cells developed severe clinical GVHD. The majority of the latter recipients died within 12 days after BM transplantation, and 95% died within 61 days. Although TLI protected BALB/c mice against GVHD induced by BM cells, all recipients given TLI and allogeneic spleen cells developed lethal GVHD. Thymic irradiation alone marginally prolonged skin allograft survival, and did not allow for allogeneic BM engraftment. These results suggest that TLI may be a useful regimen in clinical BM transplantation, since this form of radiotherapy is used extensively in humans and has few severe side effects.

Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis.

Recent investigations provided evidence that the sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor alpha (TNF-alpha) in several hematopoietic and nonhematopoietic cells. In this pathway, TNF-receptor interaction initiates sphingomyelin hydrolysis to ceramide by a sphingomyelinase. Ceramide acts as a second messenger stimulating a ceramide-activated serine/threonine protein kinase. The present studies show that ionizing radiation, like TNF, induces rapid sphingomyelin hydrolysis to ceramide and apoptosis in bovine aortic endothelial cells. Elevation of ceramide with exogenous ceramide analogues was sufficient for induction of apoptosis. Protein kinase C activation blocked both radiation-induced sphingomyelin hydrolysis and apoptosis, and apoptosis was restored by ceramide analogues added exogenously. Ionizing radiation acted directly on membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis enzymatically through a neutral sphingomyelinase. These studies provide the first conclusive evidence that apoptotic signaling can be generated by interaction of ionizing radiation with cellular membranes and suggest an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell kill.

Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.

The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells.

Vascular endothelial cells (ECs) seeded sparsely on extracellular matrix (ECM) will proliferate in the absence of exogenous basic fibroblast growth factor (bFGF). This ECM will also stimulate neurite outgrowth in PC12 cells in the absence of exogenous growth factors. We have previously shown that bFGF is found in subendothelial ECM (Vlodavsky, I., J. Folkman, R. Sullivan, R. Fridman, R. Ishai-Michaeli, J. Sasse, and M. Klagsburn. 1987. Proc. Natl. Acad. Sci. USA. 84:2292-2296) and in basement membranes (Folkman, J., M. Klagsburn, J. Sasse, M. Wadzinski, D. Ingber, and I. Vlodavsky. 1988. Am. J. Pathol. 130:393-400). The actual requirement of ECM-associated bFGF for the growth of ECs and differentiation of PC12 cells was shown in two ways. First, polyclonal anti-bFGF antibodies added to subendothelial ECM inhibited both EC proliferation and PC12 neurite outgrowth. Secondly, PF-HR-9 cells, which do not synthesize bFGF and which produce an ECM not permissive for EC proliferation and PC12 neurite outgrowth, were transfected with bFGF cDNA. PF-HR-9 cells transfected with bFGF, but not with the dominant selectable marker SV2-neomycin, were found to express bFGF and to produce an ECM which did support both EC proliferation and PC12 differentiation. The ECM-mediated stimulatory effects were inhibited by anti-bFGF antibodies but not by anti-nerve growth factor antibodies or nonimmune rabbit IgG. These results indicate that bFGF associated with ECM is a required ECM component for ECM-mediated cell proliferation and differentiation.

Long-term survival of skin allografts in mice treated with fractionated total lymphoid irradiation.

Treatment of recipient Balb/c mice with fractionated, high-dose total lymphoid irradiation, a procedure commonly used in the therapy of human malignant lymphomas, resulted in fivefold prolongation of the survival of C57BL/Ka skin allografts despite major histocompatibility differences between the strains (H-2d and H-2b, respectively). Infusion of 10(7) (C57BL/Ka x Balb/c)F1 bone marrow cells after total lymphoid irradiation further prolonged C57BL/Ka skin graft survival to more than 120 days. Total lymphoid irradiation may eventually prove useful in clinical organ transplantation.

Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.

Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism?

Basic fibroblast growth factor, (bFGF), promotes the formation of new blood capillaries and is sequestered and protected by binding to heparan sulfate (HS), both on the cell surface and in the extracellular matrix. Release of HS-bound bFGF by heparin-like molecules and HS-degrading enzymes (i.e., heparanase) provides a novel mechanism for regulation of the growth of capillary blood vessels in normal and pathological situations. The extracellular matrix also serves as a storage depot for other growth factors and enzymes.

Degradation of sulfated proteoglycans in the subendothelial extracellular matrix by human platelet heparitinase.

Cultured vascular and corneal endothelial cells produce an underlying extracellular matrix (ECM) which induces platelet adherence, aggregation, and release reaction. Incubation of a metabolically (35S)O = 4-labeled ECM with platelet-rich plasma or washed platelets, but not with platelet-poor plasma, resulted in degradation of its heparan sulfate-containing proteoglycans into labeled fragments four to five times smaller than intact glycosaminoglycan side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin, indicating that heparan sulfate in the ECM is susceptible to cleavage by the platelet heparitinase. This degradation required adhesion of platelets to the ECM rather than aggregation since it was not inhibited by aspirin, which prevented platelet aggregation but not adherence. The enzyme was not released during aggregation of platelets on the ECM but was readily liberated upon their exposure to thrombin. This liberation was inhibited in the presence of prostacyclin (PGI2). Isolated high molecular weight proteoglycans first released from the ECM by incubation with platelet poor plasma served as a substrate for further degradation by the platelet heparitinase, suggesting a cascade mechanism for degradation of heparan sulfate in the ECM. Heparitinase, although to a lower level, was also active when washed platelets were added on top of a confluent endothelial cell monolayer covering the (35S)O = 4-labeled ECM. It is suggested that the platelet heparitinase may be involved in the impairment of the integrity of the vessel wall and thus facilitate the extravasation of blood-borne cells.

Interferon enhances prostacyclin production by cultured vascular endothelial cells.

The effects of interferon (IFN) on the arachidonate metabolism and physiological functions of cultured endothelial cells and blood platelets have been examined. Cultured bovine aortic endothelial cells were found to be sensitive to the antiviral and antiproliferative activities of human leukocyte (alpha) IFN and to increase their capacity to synthesize prostacyclin (PGI2) upon exposure to IFN. Several observations indicate that IFN stimulates PGI2 synthesis at the level of the enzymes phospholipase A2 and cyclooxygenase: (a) PGI2 production was dependent upon the supply of exogenous arachidonic acid or the liberation of endogenous cellular arachidonate by ionophore A23187, but was not observed when IFN-treated cells were exposed to the endoperoxide prostaglandin H2. (b) IFN had no effect on the spontaneous release of PGI2 into the culture medium during the incubation period (24-72 h). (c) The stimulatory effect of IFN on PGI2 production was inhibited by both glucocorticoids and indomethacin. The effect of IFN on platelet prostaglandin metabolism was also investigated. Incubation of platelet-rich plasma with IFN had no effect on platelet aggregation and thromboxane A2 production. The biological significance of the findings presented in this paper may be considered in view of the protective role of PGI2 in the vessel wall and the fact that infection with certain viruses induces endothelial damage both in man and experimental animal models.

Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.

Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.

Long term effects of radiation of T and B lymphocytes in peripheral blood of patients with Hodgkin's disease.

Total lymphocyte counts, and the percentage of T and B lymphocytes and monocytes in untreated patients with Hodgkin's disease were not significantly different from those observed in normal donors. At the completion of radiotherapy, the mean total lymphocyte count of 503/mm3 was 4 SD below the mean for normal controls. Although a group of 26 patients in continuous complete remission from 12 to 111 mo after radiation treatment regained normal total numbers of lymphocytes and monocytes, they exhibited a striking T lymphocytopenia and B lymphocytosis. Concomitantly, there was a significant increase of null (neither T nor B) lymphocytes. The response of peripheral blood lymphocytes to phytohemagglutinin, concanavalin A, and tetanus toxoid before treatment was significantly impaired. 1-10 yr after completion of treatment there seemed to be little or no recovery of these responses. The capacity of peripheral blood lymphocytes to respond to allo-antigens on foreign lymphocytes in vitro (mixed lymphocyte reaction) was normal in nine untreated patients. However, the mixed lymphocyte reaction was markedly impaired during the first 2 yr after treatment. There was a partial and progressive restoration of the mixed lymphocyte reaction during the next 3 yr, and normal responses were observed in patients in continuous complete remission for 5 yr or more. The in vivo response to dinitrochlorobenzene was also examined. 88% (15/17) of patients initially sensitive to dinitrochlorobenzene were anergic to the allergen at the completion of a course of radiotherapy, but nine of these regained their hypersensitivity response during the 1st yr after treatment. This data suggests that there is a sustained alteration in both the number and function of circulating T cells after radiation therapy in patients with Hodgkin's disease which may persist for as long as 10 yr after treatment. The restoration of cell mediated immune functions after radiotherapy is time dependent and its kinetics may differ for various T-cell functions. The implications of these findings with respect to the state of immunological competence after radiotherapy are discussed.

Altered expression of the retinoblastoma gene product: prognostic indicator in bladder cancer.

BACKGROUND: It has been reported that 50%-70% of patients with bladder cancer experience recurrence after initial successful treatment and about 10%-20% of these patients die of the disease. Despite precise pathologic staging and grading, we are unable to predict clinical outcome in all patients. The retinoblastoma-susceptibility (RB) gene, a prototype of tumor suppressor genes, has recently been associated with development and/or progression of bladder cancer, as well as sarcoma and small-cell lung cancer. In transitional cell carcinomas of the bladder, we have observed altered expression of the Rb gene product--a nuclear phosphoprotein thought to function as a cell cycle regulator. PURPOSE: The aim of this study was to investigate the hypothesis that altered patterns of Rb expression correlate with prognosis in bladder cancer. METHODS: Expression of the RB gene was evaluated in specimens from 48 primary bladder tumors obtained by cystectomy or transurethral resection. Rb protein expression was correlated with disease outcome in these patients. Rb expression was examined by immunohistochemistry, using the mouse monoclonal antibody Rb-PMG3-245 on frozen tissue sections. Computerized image analysis was used to quantify the level of Rb protein in individual tumor cells. RESULTS: The overall 5-year disease-free survival was 66%, with a median follow-up of 42 months. Normal levels of Rb protein expression were found in 34 patients (Rb-positive group). A spectrum of altered patterns of expression from undetectable levels to heterogeneous expression, however, was observed in 14 patients (altered Rb group). Of the 38 patients with muscle-invasive tumors, 13 were categorized as having altered expression of Rb protein. Only one of 10 patients with superficial carcinomas had altered expression of Rb protein. The 5-year survival was significantly decreased in patients with altered Rb protein compared with the survival in patients with positive Rb expression (P less than .001). CONCLUSIONS: The results suggest that tumors exhibiting decreased expression of the RB gene-coded product (Rb protein) had a more aggressive biological behavior than those that expressed the Rb protein in the majority of their tumor cells. IMPLICATIONS: This study demonstrates that altered patterns of Rb protein expression may be an important prognostic variable in patients presenting with invasive bladder cancer.

Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells.

BACKGROUND: Protein kinase C (PKC) is a family of enzymes that function in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dihydrosphingosine, is a specific inhibitor of PKC and might represent a novel agent for anticancer therapy. Preclinical animal studies show that safingol alone has a minimal effect on tumor cell growth, but combining this compound with conventional chemotherapy agents dramatically potentiates their antitumor effects. It has been suggested that many chemotherapeutic agents exert their antitumor effects by inducing apoptosis. PURPOSE: We wanted to determine the extent to which safingol, alone or in combination with a standard chemotherapeutic agent (mitomycin C [MMC]), would promote apoptosis in gastric cancer cells in vitro. Furthermore, we investigated whether the induction of apoptosis in the treated cells was affected by their p53 tumor suppressor status or their drug-resistance status. METHODS: SK-GT-5 (p53-deficient and MMC-resistant) and MKN-74 (p53 wild-type and MMC-sensitive) gastric cancer cells were exposed to either no drug, safingol (50 microM) alone, MMC (5 micrograms/mL) alone, or a combination of safingol (50 microM) and MMC (5 micrograms/mL). In some experiments, cells were exposed simultaneously to safingol and the PKC activator, 3-phorbol 12-myristate 13-acetate (PMA), prior to treatment with MMC. Apoptosis was measured by two methods: 1) quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzamide trihydrochloride (Hoechst-33258), and 2) terminal deoxynucleotidyl transferase (TdT) labeling of the 3'-OH ends of DNA fragments produced in apoptotic cells. RESULTS: As determined by quantitative fluorescence microscopy, exposure of SK-GT-5 cells to safingol alone induced apoptosis in 2% +/- 1% (mean +/- SD) of the cells, and MMC alone increased that level to 18% +/- 1%. However, the combination of safingol and MMC induced apoptosis in 39% +/- 1% of the cells (P < .001, for the drug combination versus MMC alone). With MKN-74 cells, safingol alone induced apoptosis in 8% +/- 3% of the cells, whereas MMC alone induced apoptosis in 40% +/- 4% of treated cells and the combination of safingol and MMC induced apoptosis in 83% +/- 4% of the cells. Similar results were obtained with the TdT assay. Simultaneous exposure of cells to safingol and PMA abrogated the safingol-mediated enhancement of MMC-induced apoptosis. CONCLUSIONS: The PKC inhibitor safingol enhances the cytotoxic effect of the chemotherapeutic agent MMC in gastric cancer cells by promoting drug-induced apoptosis. The induction of apoptosis occurs regardless of the p53 status or the drug-resistance status of the cells.

Reversal of acquired resistance to doxorubicin in P388 murine leukemia cells by tamoxifen and other triparanol analogues.

The effects of the triparanol analogues chlorotrianisene, clomiphene, tamoxifen, 5-[p-(fluoren-9-ylidenemethyl)phenyl]-2-piperidineethanol (MDL 10393), MDL 8917v, nafoxidine, 2-[p-(6-methoxy-2-phenylinden-3-yl)phenoxy]triethylamine (U-11555A), 2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]triethylamine (U-10520A), and nitromifene, as well as triparanol itself, were studied in the P388 murine leukemia cell line and in a doxorubicin-resistant subline (P388/ADR). At noninhibitory concentrations, all the analogues increased the sensitivity of P388/ADR cells to doxorubicin but did not have such an effect on the doxorubicin-sensitive cells. Diethylstilbestrol, deacetylated cyclofenil (F6060), hexestrol, and 17 beta-estradiol did not have such an activity. The effects of tamoxifen on doxorubicin sensitivity of P388/ADR cells could not be reversed by 17 beta-estradiol. Estrogen receptors could not be demonstrated in either cell line. It is therefore suggested that the reversal of the doxorubicin-acquired resistance by the triparanol analogues is unrelated to their estrogenic or antiestrogenic activities. The possible clinical implications of these findings are discussed.

Reversal of acquired resistance to doxorubicin in P388 murine leukemia cells by perhexiline maleate.

The effects of perhexiline maleate on growth and drug sensitivity were studied in the P388 murine leukemia cell line and in an anthracycline-resistant subline (P388/ADR). At noninhibitory concentrations, perhexiline maleate markedly increased the sensitivity of P388/ADR cells to doxorubicin but did not have such an effect on anthracycline-sensitive cells. The effects of perhexiline maleate on P388/ADR cells were reversible. Perhexiline maleate also increased the accumulation of another anthracycline, daunorubicin, in P388/ADR cells but did not increase its accumulation in the anthracycline-sensitive cells. Perhexiline maleate did not affect the sensitivity of either cell line to methotrexate or to 6-mercaptopurine. However, its effects on the sensitivity and on drug accumulation of vinblastine, a drug to which P388/ADR cells are cross-resistant, were similar to those observed for the anthracyclines. Although perhexiline maleate has been reported to be a calcium antagonist in other systems, our data do not suggest that this mechanism is involved in its enhancement of the sensitivity of P388/ADR cells to doxorubicin. We suggest instead that this effect might be associated with alterations of cell lipid metabolism induced by perhexiline maleate.

Radiation-induced apoptosis of endothelial cells in the murine central nervous system: protection by fibroblast growth factor and sphingomyelinase deficiency.

Injury to the central nervous system (CNS) by ionizing radiation may be a consequence of damage to the vascular endothelium. Recent studies showed that radiation-induced apoptosis of endothelial cells in vitro and in the lung in vivo is mediated by the lipid second messenger ceramide via activation of acid sphingomyelinase (ASM). This apoptotic response to radiation can be inhibited by basic fibroblast growth factor or by genetic mutation of ASM. In the CNS, single-dose radiation has been shown to result in a 15% loss of endothelial cells within 24 h, but whether or not this loss is associated with apoptosis remains unknown. In the present studies, dose- and time-dependent induction of apoptosis was observed in the C57BL/6 mouse CNS. Apoptosis was quantified by terminal deoxynucleotidyl transferase-mediated nick end labeling, and specific endothelial apoptosis was determined by histochemical double labeling with terminal deoxynucleotidyl transferase-mediated nick end labeling and Lycopersicon esculentum lectin. Beginning at 4 h after single-dose radiation, apoptosis was ongoing for 24 h and peaked at 12 h at an incidence of 0.7-1.4% of the total cells in spinal cord sections. Up to 20% of the apoptotic cells were endothelial. This effect was also seen in multiple regions of the brain (medulla, pons, and hippocampus). A significant reduction of radiation-induced apoptosis was observed after i.v. basic fibroblast growth factor treatment (0.45-4.5 microg/mouse). Identical results were noted in C3H/HeJ mice. Furthermore, irradiated ASM knockout mice displayed as much as a 70% reduction in endothelial apoptosis. This study demonstrates that ionizing radiation induces early endothelial cell apoptosis throughout the CNS. These data are consistent with recent evidence linking radiation-induced stress with ceramide and suggest approaches to modify the apoptotic response in control of radiation toxicity in the CNS.

Lymphoma cell-mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: relationship to tumor cell metastasis.

Cloned lines of the methylcholanthrene-induced DBA/2 low-metastatic T-lymphoma Eb line and its highly metastatic variant ESb line were compared for the ability to degrade proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured endothelial cells. The ECM was metabolically labeled with Na2(35)SO4, and the tumor cell-mediated release of labeled degradation products was analyzed by gel filtration. More than 90% of the labeled material released upon incubation of ESb cells with the ECM, either when exposed or covered with vascular endothelial cells, was in the form of low-Mr, heparan sulfate-containing fragments (Mr approximately 10(4)) compared to high-Mr sulfated proteoglycans (mostly excluded from Sepharose 6B) released by incubation with the low-metastatic Eb cells. The same high- and low-Mr degradation products were obtained by incubation of the ECM with a serum-free medium conditioned by the low (Eb)- and high (ESb)-metastatic sublines, respectively. The high-Mr proteoglycans released by incubation of the ECM with Eb-conditioned medium was further degraded into Mr 10(4) glycosaminoglycan fragments upon a subsequent incubation with ESb-conditioned medium. These fragments were smaller than glycosaminoglycan side chains released by treatment of the ECM with papain or alkaline borohydride, suggesting an ESb-specific endoglycosidase activity. The higher ability of the ESb over the Eb cells to solubilize the glycosaminoglycan scaffolding of the sub-endothelial ECM may, among other properties, facilitate their hematogenous dissemination and extravasation.

Autocrine effects of fibroblast growth factor in repair of radiation damage in endothelial cells.

The study demonstrates that basic fibroblast growth factor (bFGF) serves as an inducer of radiation damage repair in bovine aortic endothelial cells (BAEC). Radiation dose-survival curves were generated with plateau-phase BAEC using culture dishes precoated with HR9-bFGF/extracellular matrix (ECM) for the postradiation colony formation assay. This natural basement membrane-like ECM is enriched with ECM-bound bFGF. Under these conditions the cells exhibited increased repair of radiation damage as compared to cells plated on top of the bFGF-free isotype of this extracellular matrix (the HR9/ECM). While the slopes of the curves did not differ significantly (Do 107 +/- 6.8 cGy on the HR9/ECM, compared to 112 +/- 1.3 cGy on the HR9-bFGF/ECM), there was a nearly complete elimination of the threshold shoulder in the curves generated on the bFGF-free HR9/ECM (Dq 29 +/- 19 cGy, compared to 174 +/- 22 cGy on the HR9-bFGF/ECM; P less than 0.05). Delayed plating experiments, in which the cells were irradiated under bFGF-free conditions (while adherent as contact-inhibited monolayers to the HR9/ECM in bFGF-free medium) and maintained after irradiation in the same culture for various periods of time, showed that the cells performed repair of potentially lethal damage (PLDR) and restored clonogenic ability, with a 24 h to immediate postradiation recovery ratio of 3.27. This expression of PLDR was inhibited by neutralizing monoclonal antibodies against bFGF, indicating that the irradiated cells secreted bFGF into their conditioned medium. Northern blot hybridization showed a 5.6-fold increase of the 3.7-kilobase species and a 4.7-fold increase of the 7.0-kilobase species of the bFGF-specific mRNA within 6 h after delivery of a single dose of 400 cGy. The data suggest that radiation induces a complete cycle of an autoregulated damage-repair pathway in BAEC, initiated by radiation-induced damage to cellular DNA and followed by stimulation of bFGF synthesis and its secretion into the medium. The newly synthesized bFGF stimulates the PLDR pathway, acting via an extracellular autocrine loop (inhibitable by specific anti-bFGF antibodies), leading to recovery of cells from radiation lesions and restoration of their clonogenic capacity.