Aaron Bendich

Constitutive aggregates of intermediate-sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate.

Abstract Cloned hepatoma cells ( 72 22 ) derived from the liver of a rat treated with the carcinogen, diethylnitrosamine, exhibit a genetically stable, large, acentric, juxtanuclear, hyaline aggregate of loosely packed intermediate-sized (7–11 nm) filaments, interspersed with variable but minor amounts of microtubules, polyribosomes and membranous structures. Immunofluorescence microscopy shows that the these filaments react specifically with antibodies to bovine prekeratin and to murine vimentin. The aggregates contain aster-like foci common to the arrangement of both tonofilament-like and vimentin-containing intermediate-sized filaments, although both filament systems show different fibrillar patterns in other cytoplasmic regions. While the cytokeratin filament system is not significantly altered during exposure to colcemid, the vimentin in the abnormal aggregate is rearranged during such treatment into extensive and complex perinuclear ‘whorls’ of filaments. Treatment of the cells with butyrate results in a markedly flattened, hepatocyte-like morphology, a reappearance of typical actin-containing ‘cables’, and a progressive disintegration of the filament aggregate concomitant with a normal display of filaments of both the cytokeratin and vimentin type. The results show that ( i ) some cells contain aggregates consisting of two different types of intermediate-sized filaments oriented onto a common focal center; ( ii ) such an abnormal filament arrangement is clonally stable; ( iii ) the vimentin-type filaments contained in such aggregates are still susceptible to the action of antimitotic drugs and can be rearranged into characteristic perinuclear whorls; and ( iv ) this abnormal aggregate of intermediate filaments can be reverted to normal patterns upon treatment of the cells with butyrate.

EFFECT OF DIMETHYL SULFOXIDE AND DIMETHYLFORMAMIDE ON THE GROWTH AND MORPHOLOGY OF TUMOR CELLS

The morphological pattern in which cells grow in culture has often been taken as an indication either of their malignancy or nontumorigenicity. Unlike their normal counterpart, tumor cells grow in disarray in culture, piling up and migrating past each other in a manner that resembles their behavior in tumors. We have been interested to see whether reversal of the pattern of growth back to that characteristic of nontumor cells could be achieved, and whether this would also be accompanied by loss of malignancy. The reported decrease in the malignancy of Friend erythroleukemic cells and the marked enhancement of their differentiation along the erythroid pathway after their treatment with the simple solvent dimethyl sulfoxide (DMSO)-3 stimulated our study of the effect of this agent. and of the closely related solvent dimethylformamide (DMF), on various malignant and normal cell lines used in our laboratory. An alteration in the pattern of growth of tumor cells resulted after treatment with these agents. This effect is mimicked by that of the totally unrelated agent 5bromodeoxyuridine (BrdU).4-6

DNA synthesizing activity in normal human sperm: location and characterization of the endogenous reaction.

Abstract Chromatin-associated complexes have been purified from membrane-free nuclei of normal human sperm heads. They band at a sucrose buoyant density of 1.15–1.19 g/ml and contain macromolecular components which catalyze the endogenous synthesis of DNA in a reaction partially sensitive to ribonuclease pretreatment. The sedimentation rate and buoyant density of the purified reaction product, still attached to its endogenous template, demonstrate that the [3H]DNA is associated with a high molecular weight RNA, which may be serving as both template and primer.

Information Transfer and Sperm Uptake by Mammalian Somatic Cells

Publisher Summary This chapter describes that sperm are endowed by their specific differentiation to interact with and to deliver their genetic components into ova for subsequent activation and function. Whether analogous genetic information can be transferred by sperm to mammalian somatic cells is unknown. The chapter describes that mammalian sperm interact with mammalian somatic cells as evidenced by actual penetration, the appearance of changes in morphology and growth characteristics, and the production of new gene products. These changes may be consequences of the direct acquisition and genetic expression of either the sperm chromatin itself or the reverse transcriptase containing complex within the sperm heads. The interaction of sperm with somatic cells that leads to fetal antigen expression and to changes resembling oncogenic transformation may involve a recapitulation, at least in part, of the program of postfertilization gene expression. The normal function of sperm is fertilization; the presence of its various components in somatic cells might very well induce changes analogous to those that occur in embryological and fetal development. It has been estimated that the amount of unique-sequence DNA that is transcribed in the rabbit blastocyst but no longer in the midgestation embryo is enough to specify some 23,000 different proteins; the corresponding difference between normal and virus-transformed mouse cells is regarded as sufficient to specify some 200,000 different proteins. To sort out the many steps involved in embryogenesis, fetal development, and oncogenesis in the face of such complexity is indeed a prodigious task.

IN VIVO-IN VITRO RAT LIVER CARCINOGENESIS: MODIFICATIONS IN PROTEIN SYNTHESIS AND ULTRASTRUCTURE*

Different experimental approaches have been used to study the progression of cellular events that accompany the development of hepatic neoplasia. Following the administration of carcinogens to the intact animal, tissues can be excised at predetermined times and alterations evaluated by morphological, histochemical, and immunological examination of either tissue sections or homogenates. Since liver tissue is both structurally and functionally heterogeneous, the identification and isolation of specific cellular populations becomes essential in the critical evaluation of any neoplastic changes. That the parenchymal liver cells engaged in the synthesis of organ-specific proteins are morphologically, biochemically and functionally different from others within a given liver lobe and appear to be randomly distributed3 is reflective of this organizational heterogeneity. Hepatocytes located in the peripheral zone differ in function and morphology from those in the centrolobular zone and might thus be expected to react differently to various exogenous stimuli. It has indeed been difficult to determine whether a certain subset of parenchymal hepatocytes is responsible for the synthesis of a particular plasma protein or whether, in fact, all are capable of synthesizing these proteins provided they are turned on by an appropriate signal. The observations that only 35 percent of hepatocytes are synthesizing plasma proteins in vivo at any given time thus requires further elucidation. This ambiguity in the determination of a specific parenchymal cell function may well reflect the complexities imposed on in vivo studies by such factors as cellular heterogeneity and homeostatic mechanisms. The response of a given cell population to exogenous agents, such as carcinogens, under in vitro conditions might provide information heretofore not attainable in studies with intact animals. Long-term in vitro studies therefore could have a distinct advantage by allowing for the direct examination, over extended periods of time, of cells maintained under controlled conditions. Some dificulties encountered with cultured cells however are l) the limited ability of these cells to activate various carcinogens as efficiently as in vivo, and 2) the relative infrequency with which useful normal liver lines have been established for carcinogenesis studies. Therefore, in order to avoid the problems inherent in the activation of carcinogens by cells in culture, we selected a combined in vivo-in vitro approach to the study of hepatocellular carcinogenesis in which metabolic activation of the carcinogen occurred in vivo. Our model system employed Wistar rats, to which diethylnitrosamine (DENA)

Differential association of fetal antigen with hepatoma tissue grown in vivo and in vitro.

Abstract Production of two distinct fetal antigens, alpha-fetoprotein (AFP) and γ-fetal antigen (γ-FA) was associated with growth of the BW7756 mouse hepatoma in vivo . Synthesis of AFP, but not γ-FA, continued during in vitro propagation of the tumor cells. After re-inoculation of these cultured hepatoma cells into inbred mice, both AFP and γ-FA could be detected again in the growing tumor tissue and in the sera of tumor-bearing mice. It is evident that different growth conditions effect synthesis of these two tumor-associated antigens.

Isolation of a nuclear DNA synthesizing complex from human sperm.

Summary A simplified procedure is described for isolating a human sperm nuclear DNA-synthesizing complex which does not involve the use of detergent or necessitate the prior purification of membrane-free nuclei. Selective disruption of sperm heads is accomplished by the use of dithiothreitol, trypsin, and deoxyribonuclease. The particulate complex is freed of soluble components by sedimentation through a solution of 19% (w/v) Metrizamide and further purified by buoyant density centrifugation. The characteristics of this endogenously templated nuclear DNA polymerase differ from other sperm-associated polymerases that have been reported.

A fetal antigen in a mouse fibrosarcoma with possible cross-reactivity with an adult mouse skin component

Abstract A late gestation—neonatal phase antigen has been found in a mouse fibrosarcoma. The cross-reacting antigen of tumor, fetal and neonatal mice possessed gamma-mobility upon immunoelectrophoresis in 2% agar at pH 8.6 and was undetectable in extracts of normal adult internal organs. A related, unstable, constituent was detected in normal adult mouse skin.

In vivo initiated rat liver carcinogenesis studied in vitro; formation of alcoholic hyaline-type bodies

Epithelial liver cells isolated from rats after oral diethylnitrosamine administration were established in culture. When propagated in vitro for 2--3 months, over half the cells contained acentric nuclei and large juxtanuclear inclusions resembling the hyaline deposits (Mallory bodies) in cirrhotic livers of alcoholics. The morphological changes and disarrayed filaments in these bodies were retained on serial passages for many months. Cells inoculated into nude mice or newborn rats produced carcinomas from which cells with these abnormalities were reestablished in continuous culture.

In Vitro consequences of sperm-somatic cell interactions

Abstract Following interaction with rat spermatozoa and subsequent proliferation in vitro , Chinese hamster lung fibroblasts synthesized fetal antigen and grew in semisolid agar suspension culture but were not tumorigenic in nude mice. Control hamster cells were consistently negative for these properties even after long-term cultivation in liquid medium. Admixture with spermatozoa was also found to induce marked multinucleation and to enhance the cloning efficiency of human tumor (HeLa) cells in soft agar culture. Sperm-mediated induction of fetal antigen synthesis and anchorage-independent growth by cultured mammalian somatic cells may thus represent an early stage in the multi-step sequence leading to malignant transformation. Sperm-somatic cell culture systems may provide a convenient model system for the study of cell-cell interactions similar to those which may normally occur in vivo .