Faculty Bibliography

Established 72/22 rat hepatic epithelial tumor cells, which possess intracellular aggregates of intermediate-sized filaments resembling Mallory-body-like inclusions, were used to assess changes in tumor cell growth and morphology associated with exposure to contact-inhibitory factor (CIF). CIF reduced 72/22 proliferative rate, increased mean population doubling time by 42%, lowered culture saturation densities to 34-50% of control values and inhibited formation of dense foci. These proliferative changes were due to an apparent prolongation of the G1 phase of the cell cycle during the period of CIF exposure. CIF concomitantly induced a marked increase (by 70%) in cell spreading and loss of both the usual tight (epithelioid) cell juxtaposition and typical ordered colony structure characteristic of untreated populations. However, CIF exposure failed to achieve complete cytoarchitectural 'normalization' in 72/22 cells (i.e., dispersal of the Mallory-body-like aggregate of intermediate filaments and restoration of a more typical hepatocytic phenotype). Most obvious was a reduction in the integrity of the peripheral band of microfilaments (a structure involved in the maintenance of epithelial cell shape) and a decrease in the content of desmoplakin (a protein component of desmosomal plaques). Changes in these major structural elements appear to be critical events in development of the pleomorphic phenotype and reduced substratum adhesiveness observed during treatment. CIF-related fragmentation of peripheral band structures was not reflected in changes in either the total cellular or cytoskeletal-associated actin contents. The morphologic changes observed under conditions of CIF exposure closely paralleled induced decreases in the cellular content of the actin-associated membrane skeleton protein p35. These data collectively suggest that CIF may act to alter the composition of the cortical skeleton in cultured liver tumor cells

It is well recognized that physical contact between natural killer (NK) cells and tumor targets is necessary for cell lysis. Therefore, any modulation of the tumor cell surface that alters intercellular contact could affect NK cell cytotoxicity. To examine this hypothesis, a contact inhibitory factor (CIF), which had been shown to restore contact inhibition of growth to several malignant cell lines was tested for its ability to render such cells immune to recognition by NK cells. When three NK-sensitive melanoma and two NK-sensitive colon carcinoma targets were cultured with CIF, they did not only change morphologically, but also showed a 70% to 95% reduction in their sensitivity to lysis by NK cells. In addition, K562 cells, which grow in suspension and do not permit a morphologic evaluation of the CIF effect, also became resistant to lysis by NK cells after culture with CIF. CIF did not reduce the viability nor the cytotoxicity of NK cells. CIF did not contain interferon nor did the CIF-treated targets induce the production of interferon during the cytotoxicity assay. It is concluded that restoration of contact inhibition of growth and resistance to NK cell lysis are cell surface phenomena that may run in parallel

Conditioned medium (CM) from confluent cultures of the contact-inhibited hamster melanocytic cell line, FF, contains a biologic activity, contact inhibitory factor (CIF), which reversibly restores density-dependent growth to melanoma cells. When a hydrophobic affinity-concentrated extract of CIF-containing CM was incorporated in agarose at a concentration of 1000 micrograms protein/ml, it restored anchorage-dependent growth to RPMI 1846 hamster melanoma cells. Colony-forming efficiency in CIF-treated wells decreased to 5% from levels of 51.5% in controls prepared with regular growth medium. In addition, CIF-containing CM restored serum-dependent growth to RPMI 1846 cells, markedly restricting proliferation in 1% calf serum-containing medium. Control cultures containing 1% calf serum and either complete growth medium or CM from the non-contact-inhibited hamster melanoma line itself, supported proliferation of RPMI 1846 cells to levels 3.9 X and 3.7 X that of CIF-treated cultures, respectively. CIF is the first factor derived from contact-inhibited mammalian cell cultures that has been shown to restore density-, anchorage-, and serum-dependent growth to malignant melanoma cells

Contact inhibitory factor (CIF) is a growth inhibitor obtained from conditioned culture medium of a contact-inhibited line of hamster melanocytic cells, which reversibly restores density-, anchorage-, and serum-dependent growth to melanoma cells. The usefulness of liposomes as carriers for CIF was investigated in vitro. The stability of liposomes prepared both with and without CIF was demonstrated by measuring the rate of efflux of a K2CrO4 marker. Anionic multilamellar lipid vesicles (7 phosphatidylcholine:2 dicetyl phosphate:1 cholesterol) prepared with CIF-containing material and separated from unentrapped CIF by gel filtration on Sepharose 2B, showed retarded leakage of a K2CrO4 marker (half-efflux at 77 h) when compared with identical liposomes lacking CIF (half-efflux at 40 h). When added to subconfluent cultures of hamster melanoma cells, liposome-entrapped CIF restored contact-inhibited growth. Compared with aqueous solutions of CIF, liposome-CIF effects were characterized by longer latency and more sustained duration. The ability of CIF-bearing liposomes to effectively restore density-dependent growth in vitro should facilitate in vivo studies of the effects of this potent growth inhibitor on melanoma and other neoplasms

Most vitiligo sera contain antibodies to surface antigens on pigmented human melanocytes but not to human or mouse amelanotic melanoma cells. A density-dependent line of hamster amelanotic melanocytic cells (FF) produces a diffusible factor (CIF) which restores contact inhibition of growth as well as several other normal phenotypic characteristics to hamster, murine, and human melanoma cells. The ability of CIF to induce the expression of a phenotypic characteristic of pigmented human melanocytic cells, i.e., the vitiligo-related surface antigens, on hamster and mouse amelanotic melanoma cells was investigated. Vitiligo and normal sera were reacted with CIF-treated and untreated hamster and mouse amelanotic melanoma cells for both indirect-immunofluorescence assays and ELISA. Immunofluorescence testing showed that about 80% of hamster and mouse melanoma cells had pigment-cell antigens (in the absence of pigmentation) in a granular surface pattern after, but not prior to, CIF-induced morphologic reversion and confluent growth. Less than 5% of the control hamster and mouse melanoma cells expressed such antigens at confluence. These results were confirmed by ELISA. Metabolic-labeling studies with 35S-methionine showed that the vitiligo antigens were synthesized by the CIF-treated melanoma cells. The slowing of melanoma cell proliferation in isoleucine-deficient medium failed to elicit the expression of vitiligo antigens. Since antigen appearance following phenotypic reversion occurred without pigment induction, it is concluded that vitiligo-related surface antigens and pigmentation are distinct aspects of a differentiated function which may be non-coordinately expressed. The expression of pigment-cell differentiation antigens on amelanotic melanoma cells is an additional feature of the pleiotypic trans-species response to CIF