Middle Ear "Adenoma": a Neuroendocrine Tumor with Predominant L Cell Differentiation.

This morphological and immunohistochemical study demonstrates that tumors currently known as "middle ear adenomas" are truly well-differentiated epithelial neuroendocrine tumors (NETs) composed of cells comparable to normal intestinal L cells, and therefore, these tumors resemble hindgut NETs. These tumors show consistent expression of glucagon, pancreatic polypeptide, PYY, and the transcription factor SATB2, as well as generic neuroendocrine markers and keratins. The same L cell markers are expressed by cells within the normal middle ear epithelium. These markers define a valuable immunohistochemical profile that can be used for differential diagnosis of middle ear neoplasms, particularly in distinguishing epithelial NETs from paragangliomas. The discovery of neuroendocrine cells expressing the same markers in non-neoplastic middle ear mucosa opens new areas of investigation into the physiology of the normal middle ear and the pathophysiology of middle ear disorders.

Tumorigenicity of intraspecific somatic cell hybrids in nude mice.

Intraspecific somatic cell hybrids between normal mouse peripheral blood lymphocytes and a highly tumorigenic L-cell line (C1-1D) produced tumors in nude mice. While the hybrid cells were tumorigenic, the length of time necessary for tumor appearance and the size of the tumor varied. Correlation between the growth rate of the parenteral and hybrid cells in vitro or their plating efficiency in methyl cellulose with the rapidly of tumor growth in vivo was not found.

Vitamin A-induced density-dependent inhibition of L-cell proliferation.

Retinoic acid, the acid form of vitamin A, was found to have an inhibitory effect on the proliferation of L-929 mouse cells. Cultures treated with retinoic acid (5.0 mug/ml) were shown to cease proliferation at cell densities corresponding to confluent monolayers (10.0+/-1.0 X 10(4) cells/cm2). Control cultures, however, continued to proliferate and consistently reach densities two to four times higher than those of treated cultures. Viability was determined by trypan blue exclusion, and the results (80-90 percent viable) excluded cytotoxicity as an explanation of decreased proliferation. Replenishing the medium on confluent retinoic acid-treated cultures failed to stimulate further proliferation, while control cells continued to grow exponentially with each medium change. Therefore, the cessation of cell proliferation at confluence did not result from medium depletion. Studies of cell growth after seeding at relatively low cell densities have indicated that retinoic acid-treated cultures had greater nutritional requirements than did control cultures. Cell-cloning experiments have shown that DNA synthesis was not blocked, since clones formed by cells seeded with retinoic acid contained an average of 27 cells after 9 days of incubation (indicating between four and five cell divisions). However, clones developing from treated cells had fewer cells per clone (27 vs. 54) and were less dense than control clones. These data suggested the restoration of contact inhibition (topoinhibition) to L-929 cells treated with retinoic acid.

Effects of tocopherol (vitamin E) acid succinate on morphological alterations and growth inhibition in melanoma cells in culture.

The effects of various forms of tocopherol (vitamin E) on the growth and differentiation of mouse melanoma (B-16) and mouse fibroblast (L-cells) cells in culture were studied. D-alpha-tocopherol acid succinate induced morphological alterations and growth inhibition in melanoma cells. When vitamin E acid succinate was removed 4 days after treatment, the above changes remained irreversible for a period of 24 hr, after which resistant cells and partially affected cells renewed cell division and eventually reached confluency. The relative efficacy of D and DL forms of vitamin E acid succinate remains to be evaluated. However, other forms of vitamin E such as DL-alpha-tocopherol free alcohol, Aquasol DL-alpha-tocopherol acetate, DL-alpha-tocopherol nicotinate, or sodium succinate with an equivalent volume of ethanol, at similar concentrations, were ineffective. Vitamin E acid succinate at similar concentrations did not induce morphological changes in fibroblasts. Melanoma cells were about 2-fold more sensitive to vitamin E acid succinate than were fibroblasts for the criterion of growth inhibition. Vitamin E acid succinate-induced morphological changes and growth inhibition in melanoma cells were expressed in hormone-supplemented serum-free medium, but the concentration requirement was about 5 times less than that needed in serum-supplemented medium. Although cyclic adenosine 3': 5'-monophosphate-stimulating agents are known to cause growth inhibition and morphological changes in melanoma cells in culture, vitamin E acid succinate-induced morphological alterations in melanoma cells are no mediated by a rise in cellular cyclic adenosine 3':5'-monophosphate. Ethanol was sufficient to increase the melanin content in melanoma cells. These data show that vitamin E acid succinate may be a potentially useful tumor therapeutic agent.

Mechanism of action of inosine dialdehyde (NSC 118994) in the inhibition of proliferation of tumor cells in culture.

Inosine dialdehyde (INOX), the periodate oxidation product of inosine, inhibited the proliferation of various tumor cell lines in suspension culture in a concentration-dependent manner. A concentration of about 1 mM was required to completely inhibit the proliferation of Novikoff rat hepatoma and mouse L-cells, whereas about 0.1 mM completely inhibited the proliferation of L1210 and P388 mouse leukemia and Chinese hamster ovary cells. INOX inhibited in a similar time- and concentration-dependent manner the synthesis of protein, RNA, and DNA, as measured by the incorporation of labeled amino acid, uridine, and thymidine, into acid-insoluble material, without significantly affecting the incorporation of these precursors into the acid-soluble pool. Flow microfluorometric analyses showed that many of the INOX-treated cells became arrested in G2 + M. The results are consistent with the view that INOX affects multiple metabolic steps. The effects of INOX were quite different from those caused by typical inhibitors of ribonucleotide reductase, hydroxyurea, and 2,3-dihydro-1H-pyrazolo(2,3-a)imidazole, which very rapidly inhibited DNA synthesis and caused arrest of the cells in G1, with minimal effects on RNA and protein synthesis.

Preferential uptake of cytotoxic porphyrins from hematoporphyrin derivative in murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells.

Photodynamically induced loss of clonogenicity of murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells was determined with two different assays. It appeared that the loss of clonogenicity was much higher when 20 cells/cm2 were incubated with hematoporphyrin derivative (HPD) and illuminated, than when confluent cell layers were incubated with the same amount of HPD and illuminated prior to plating out. This dependency of cell killing on the experimental protocol was also observed when protoporphyrin (90-95% pure) was used as photosensitizer, but not when the cells were photodynamically treated with rose bengal or exposed to mitomycin C. Further, when cell layers were incubated with the residual solution that remained after the previous incubation of a confluent cell layer with HPD, illumination of these layers appeared to be almost non-toxic, although the overall porphyrin concentration in the residual solution was only slightly lower than in HPD. These results indicate that the porphyrins, responsible for loss of clonogenicity, are present in relatively small amounts in HPD and unpurified protoporphyrin and are preferentially taken up by the cells. Although 2-aminoisobutyric acid transport and DNA synthesis are among the most photosensitive targets with HPD, photodynamic treatment of L929 cells with the residual solution did not result in inhibition of the transport system and DNA synthesis. In contrast, the K+ content of the cells still decreased considerably, when utilizing the porphyrins, remaining in the residual solution as sensitizer. This indicates that under the present experimental conditions the disturbance of the membrane barrier function does not contribute to loss of clonogenicity of these cells and, moreover, that the photodynamically induced K+ leakage is caused by a component of HPD other than inhibition of 2-aminoisobutyric acid transport and DNA synthesis.

Development of metastatic tumors in athymic (nude) mice from LM cells grown in vitro.

The capacity of LM cells to initiate tumor formation in vivo was investigated in athymic (nude) BALB/c mice. Tumor cells inoculated s.c. over the left thighs of mice formed large, grossly visible tumors within 2 weeks postinoculation. Tumor doubling times were slightly longer than doubling times of LM cells in suspension culture. Histological examination of necropsied mice revealed that tumor cells were invasive into host tissue and formed numerous bizarre mitotic figures. The tumors were classified a fibrosarcoma-like tumors. Metastasis to the lungs was observed in nude mice inoculated with a variety of tumor cells. LM cells cultured in the presence of choline analogues (N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine) had grossly altered membrane lipid compositions. However, all formed tumors in the nude mice with tumor volume doubling times similar to that of LM cells grown in choline medium. LM cells grown with 10% calf serum had doubling times about 42% shorter. LM fibroblasts injected into nude mice provide a model system for investigation of tumor biology related to metastasis formation.

Adhesion of leptospires to mouse fibroblasts (L929) and its enhancement by specific antibody.

The adhesion of leptospires (Leptospira interrogans serovar. copenhageni L45) to mouse L-cells was studied by microscopic observations. Within 3 h of infection of monolayers many leptospires adhered to 95-100% of the cells, and intracellular leptospires were demonstrated by electron microscopy. No specific site of attachment on the cells or the leptospires was observed. Avirulent or dead leptospires adhered poorly but attachment of the saprophytic leptospire L. biflexa serovar. patoc occurred on cell and glass surfaces. After adhesion, microvilli on the cell surfaces disappeared within 6 h of infection and cell damage was observed after 12 h. The adhesion was greatly enhanced by the presence of specific antiserum at a subagglutinating concentration. No direct penetration by leptospires of the host cells was observed with transmission and scanning electron microscopy. It appears that (1) adhesion of leptospires to L-cells precedes cell damage, and (2) leptospires may enter cells either through damaged membranes, or by a phagocytosis-like mechanism.

[Cytotolerance of bioceramics in cell culture studies]. / Cytotolerance biokeramiky sledovaná v bunecných kulturách.

Biological effects of ceramic implant material of Czechoslovak make prepared on the basis of alumina were studied here. The material was proved to be quite inert both toward epithelioid Hep-2 cells and toward L-line fibroblastoid cells in experiments with nonspecific application, i.e., in cell cultures.

Evaluation of the mutagenic effects of SV40 in mouse, hamster, and mouse-human hybrid cells.

We have examined the ability of SV40 to induce changes in drug or temperature resistance in mouse, hamster, and mouse-human hybrid cells. SV40 induced a substantial increase of cells resistant to 5-bromodeoxyuridine + trifluorothymidine in Balb/c 3T3 cells and induced an increase of hybrid cells resistant to 6-thioguanine. SV40 was found to be nonmutagenic or weakly mutagenic in other test systems. The 3T3 cells were T-antigen positive, exhibited a marked reduction in TK activity, were heterogeneous for [3H]BrdU incorporation by autoradiography, and exhibited instability of the drug-resistance phenotype, suggesting that SV40 may be inducing resistance by an epigenetic process. SV40-induced 6-thioguanine resistance in the hybrids appears to occur predominantly by chromosome loss.

Modulation of the malignant phenotype with the urokinase-type plasminogen activator and the type I plasminogen activator inhibitor.

Gene transfer techniques were utilized to evaluate the role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in enhancing or preventing the expression of the invasive malignant phenotype, respectively. Mouse L-cell transfectants expressing human uPA or human PAI-1 as well as mouse B16 transfectants expressing mouse uPA or human PAI-1 were generated. These transfectants were tested using a variety of experimental methods including smooth muscle cell matrix solubilization in vitro, lung colony formation in vivo and co-cultures of antagonist-expressing cells in vitro. Results from these studies provide direct evidence for an enhancing role of uPA in malignant invasion and experimental metastasis and for a modulatory role of PAI-1 in tumor cell-mediated breakdown of extracellular matrices.

Effects of daunomycin and radiation on cell-survival and repair of DNA single-strand breaks.

The combined action of Daunomycin and irradiation was investigated using mouse L-929 cells in culture. Survival of cells was measured with the colony assay. Sedimentation in alkaline sucrose gradients was used to study repair of DNA single-strand breaks (SSB) in the presence of various concentrations of Daunomycin. A small increase in radio-sensitivity, as measured by decreasing Do, was obtained for doses of Daunomycin that are considerably toxic to the cells (0.1 microgram/ml). However, the Dq values remained constant even at high concentrations indicating that Daunomycin does not interfere with recovery processes. The rate of rejoining of SSB remained constant up to 1.0 microgram/ml, whereas concentrations of Daunomycin as high as 10 microgram/ml reduced the velocity of repair by a factor of 13. Our data show that concentrations of Daunomycin similar to those required for other DNA-binding drugs are required to inhibit SSB repair. For clinical purposes, no increase in tumour-killing efficiency may be expected from a combined treatment with Daunomycin and radiation.