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Although stem cells succumbing to reproductive death are assumed to be the single relevant targets in radiation tissue damage, recent studies showed intestinal stem cell damage is conditionally linked to crypt endothelial apoptosis, defining a two-target model. Here we report that when mouse intestines were protected against microvascular apoptosis, radiation switched as the dose escalated to a previously unrecognized crypt stem cell target, activating ceramide synthase-mediated apoptosis to initiate intestinal damage. Whereas ataxia telangiectasia-mutated (ATM) kinase normally represses ceramide synthase, its derepression in Atm(-/-) mice increased crypt stem cell radiosensitivity 3.7-fold without sensitizing the microvascular response. Discovery of this intestinal radiosensitivity mechanism allowed design of an antisense Atm oligonucleotide treatment which phenocopied the Atm(-/-) mouse, reordering ceramide synthase-mediated stem cell death to become the first-line gastrointestinal response of wild-type littermates. These experiments indicate that tissues operate multiple potential targets activated consecutively according to their inherent radiosensitivities that may be reordered therapeutically to control radiation tissue responses.

Previous studies provided evidence that MCA/129 fibrosarcomas and B16 melanomas grow 2- to 4-fold faster in acid sphingomyelinase (asmase)-deficient mice than in asmase(+/+) littermates and are resistant to single-dose irradiation due to inability to mount an apoptotic response in tumor microvascular endothelium. However, others postulated the differences might be associated with a host antitumor immune response in asmase(+/+) mice that is not expressed in asmase(-/-) mice due to phenotypic deficiency in antitumor immunity. The present studies demonstrate that none of the tumor-host combinations displayed the classic criteria of an immunogenic tumor because they lacked endotumoral or peritumoral infiltrates almost entirely. Furthermore, neither MCA/129 fibrosarcoma nor B16 melanoma tumors showed differences in growth or radioresponsiveness when implanted into mutant mouse models (Rag(-/-) and MEF(-/-)) lacking functional immune cell [natural killer (NK), NK-T, T, and B cells] populations. Additionally, development and function of B-, T-, and NK-cell populations in asmase(-/-) mice were normal, indistinguishable from their wild-type littermates. These data provide definitive evidence that MCA/129 fibrosarcomas and B16F1 melanomas do not elicit a host immune response in wild-type mice and that the asmase(-/-) phenotype is not deficient in antitumor immunity, supporting the notion that the patterns of tumors growth and radiation response are conditionally linked to the ability of the tumor endothelium to undergo ASMase-mediated apoptosis.

Cell lines derived from human prostate cancer are regarded as relatively resistant to both radiation-induced clonogenic death and apoptosis. Here we attempted to modulate the response of LNCaP prostate cancer cells to radiation therapy (XRT) by pretreatment with 12-O-tetradecanoylphorbol acetate (TPA), a known apoptogenic agent in LNCaP cells. Using plateau-phase cultures, we investigated the response of these cells to XRT, TPA, and a combination of XRT and TPA. LNCaP irradiation did not result in ceramide generation or apoptosis. However, pretreatment with TPA enabled XRT to generate ceramide via activation of the enzyme ceramide synthase and signal apoptosis. Apoptosis was abrogated by the competitive inhibitor of ceramide synthase, fumonisin B1. Furthermore, when transplanted orthotopically into the prostate of nude mice, LNCaP cells produced tumors that recapitulated the responses of LNCaP cells in vitro. XRT or TPA failed to signal apoptosis in LNCaP tumors, whereas a combination of the two resulted in substantial (20-25%) apoptosis within 24 h. There was an additional benefit associated with this regimen because TPA pretreatment protected the adjacent rectum from radiation-induced apoptosis. This represents the first description of signaling-based therapy designed to overcome one form of radiation resistance expressed preferentially in LNCaP human prostate cancer cells.

It has been reported that aortic homografts that have been cryopreserved before transplantation remain viable longer as an allograft than tissue stored at 4 degrees C in an antibiotic solution. In the present study, we tested the hypothesis that storage of cardiac valve tissue by cryopreservation or by antibiotic preservation may alter the metabolic status of the tissue. Initially, we collected aortic valves composed of cardiac tissue, aortic root, and valvular tissue from cadaver donors. These specimens were divided into three equal portions, and one portion was analyzed before storage while the other two parts were stored for 3 weeks at either 4 degrees C in an antibiotic solution or at -196 degrees C in liquid nitrogen. All specimens were examined with regard to the following parameters: tissue structure, tissue viability, cell proliferative capacity, metabolic function, and identification of cell-specific antigens. We found no significant alterations in the structure of any of the three tissue components after antibiotic preservation or cryopreservation; however, cell viability and cell number were decreased in all three groups. All tissue samples grew in culture before storage. When we compared activities of the following organellar marker enzymes--lysosomal acid lipase, plasma membrane 5' nucleotidase, mitochondrial cytochrome oxidase, and microsomal neutral alpha-glucosidase--we observed no major differences between tissues stored by either technique. In addition, we observed no loss of enzymic activity as a result of storage. Finally, when cell lines isolated from each tissue specimen were incubated with monoclonal antibodies against cell-specific antigens in an immunoperoxidase assay, all the cell cultures proved to be endothelial cells. These results suggest that although cardiac valve tissue stored by cryopreservation or by antibiotic preservation retained its normal structure and metabolic capabilities, both storage techniques produced significant decreases in cell numbers and viability. However, only endothelial cells from tissue stored by cryopreservation retained the capacity to proliferate in vitro. These findings have important implications for the function of aortic homografts transplanted after storage.

Background: Class I and Class II histocompatibility leukocyte antigens (HLA) play an important role in the antigenic recognition and target cell killing by T-lymphocytes. Their expression and modulation with gamma interferon on human soft tissue sarcomas were investigated., Methods: The phenotypic expressions of Class I and Class II HLA were determined by avidin-biotin immunoperoxidase staining using two monoclonal antibodies W6/32 and MEL3, respectively., Results: The present study showed that soft tissue sarcomas frequently had demonstrable Class I HLA and less-frequently expressed Class II HLA: The staining for Class I HLA was more diffuse, and the staining for Class II HLA was generally patchy in appearance. The expressions of two antigens on cultured sarcoma cells were found in accordance with the findings of sarcoma tumors. The expression of Class I antigen was enhanced, and Class II was induced in two cell lines by gamma interferon. The in vitro modulation of HLA with gamma interferon was reversible. Gamma interferon at the testing dose did not have cytotoxic or antiproliferative effects on either cell lines., Conclusions: Through the modulation of HLA on soft tissue sarcomas, gamma interferon may play a role in the clinical management of sarcomas.

HT-29-15 is an IgG1 monoclonal antibody reacting with a neuraminidase-sensitive determinant on a cell-surface antigen (molecular weight, 200,000 daltons) present on the colon cancer cell line HT-29. HT-29-15 was selected for a tumor localization study because the antigen was shown to be present, by immunohistochemical staining, in a high percentage of primary and metastatic colorectal cancers. HT-29-15 labeled with iodine 131 was given intravenously over a dose range of 0.2 to 10.0 mg to 23 patients with colorectal cancer. No significant toxicity was seen. Imaging of hepatic metastases was successful from days 5 to 7. Analysis of tissue radioactivity by biopsy showed that the tumor-liver ratio increased from day 1 to day 7, suggesting more rapid clearance of antibody from normal tissue than from tumor. Thus, tissue biopsy specimens and scintigraphy have shown that imaging of metastatic colorectal cancer is possible with monoclonal antibody HT-29-15. Tissue biopsy specimens are essential for demonstrating specificity of localization. Scans alone provide insufficient evidence of specific localization by monoclonal antibodies. Simultaneous infusion of a nonreactive control antibody would be necessary for specific localization to be demonstrated unequivocally.

Sera from 6 of 12 patients with paraneoplastic cerebellar degeneration (PCD) contained anti-Purkinje cell antibodies, as determined by indirect immunofluorescence on frozen sections of normal human cerebellum. Samples of cerebrospinal fluid from 2 of the patients with serum antibodies were tested, and both specimens contained anti-Purkinje cell antibody. The anti-Purkinje cell antibodies were polyclonal, fixed complement, and were present in all patients at serum dilutions of 1:1,000 or greater. Antibody activity could not be suppressed by preabsorption of sera with human or animal brain and tissue powders or with fresh crude human cerebellar extracts. No anti-Purkinje cell antibodies were detected in control sera from 167 neurologically normal cancer patients, 32 normal volunteers, 10 patients with other causes of cerebellar degeneration, or 8 patients with other paraneoplastic neurological diseases. Preliminary evidence suggests that the Purkinje antigen is a protein that is often concentrated in the periphery of the cytoplasm in disc-shaped structures. Patients with antibodies often developed signs of PCD near the time of detection of the tumor and had relentless progression of neurological disease. Patients without antibodies frequently had cancer for months to years before PCD developed, and often had spontaneous stabilization of neurological disease with time. Four patients without and 3 patients with antibodies underwent plasmapheresis without response.

Twenty-two benign pleomorphic adenomas of the major salivary glands were studied by transmission electron microscopy and immunohistochemical techniques (three cases) in order to characterize the cell types comprising the epithelial and so-called mesenchymal regions of the tumors. Light- and electron-microscopic studies showed the tumors to consist of variable mixtures of neoplastic ductular epithelial cells, rare acinar cells, and metaplastic myoepithelial cells. Many of the loosely organized "stromal cells" contained structures indicative of their myoepithelial origin, e.g., perinuclear tonofilaments, ectoplasmic actin microfilaments, and remnants of basement membrane. Polyclonal antikeratin antisera strongly stained ductular epithelial and myoepithelial cells, squamoid cell nests, and periductular myoepithelial cells, whereas myxoid and chondroid cells were less intensely stained. Monoclonal cytokeratin antibody AE1 stained only the ductular epithelial cells in both the normal glands and tumors. In contrast, S-100 protein, which is present only in scattered acinar cells and myoepithelial cells in the normal parotid gland, was found in the ductular and periductular myoepithelial cells, isolated myxoid cells, and chondroid and cartilagenous cells in the tumors. Actin was found in all the cell types of the tumor but staining was strongest in the ducts. Double immunofluorescence staining for cytokeratin and vimentin revealed coexpression of both types of intermediate filaments in occasional normal acinar and intercalated duct myoepithelial cells, and in some cells in the myxoid and chondroid regions of the tumors. In the tumors, vimentin was present in occasional periductular myoepithelial cells, stellate myxoid cells, and especially in chondroid cells and chondrocytes. Our findings indicate that benign pleomorphic adenomas of the major salivary glands are pure epithelial cell tumors. The histologic complexity of these neoplasms is due to the ability of the neoplastic ductular myoepithelial cell to modulate its morphologic appearance and intermediate filament composition, and to produce large amounts of matrix substances. We further postulate that these tumors arise from neoplastically transformed intercalated ducts.