Thrombospondin binding by human squamous carcinoma and melanoma cells: relationship to biological activity

Human squamous carcinoma cells attach and spread on thrombospondin (TSP)-coated culture dishes but exhibit significant variability among individual cell lines in their degree of responsiveness. Using a highly responsive squamous carcinoma line and a cell line which is much less responsive (as well as a human melanoma cell line which does not respond at all in the adhesion assay), we have examined binding of exogenous radiolabeled TSP. The cells which were the most responsive to TSP in the adhesion assay bound the greatest amount of radiolabeled ligand. Binding was time- and dose-dependent, saturable, inhibitable with excess unlabeled TSP, reversible, and specific. The less-responsive squamous carcinoma cells bound only 25-30% of the amount of TSP bound by the highly responsive cells while the nonresponsive melanoma cells bound less than 10% of the amount bound by the highly responsive squamous carcinoma cells. Our previous studies (J. Varani et al. (1986) Exp. Cell Res. 167, 376) have shown that the highly responsive squamous carcinoma cells also synthesized the greatest amount of TSP as indicated by biosynthetic labeling studies. The less-responsive squamous carcinoma cells were intermediate in synthetic activity and no synthetic activity was seen with the melanoma cells. These findings suggest that the amount of ligand bound may determine the degree of biological responsiveness and that endogenously synthesized TSP may be the source of that ligand.     

Phosphorylated isomers of L-dopa stimulate MSH binding capacity and responsiveness to MSH in cultured melanoma cells

L-dopa is a key metabolite in the process of melanogenesis. However, it is difficult to use in biological experiments because it is subject to auto-oxidation and relatively insoluble at neutral pH. Dopa phosphates contain phosphate ester linkages at positions 3 and/or 4 of the phenylalanine ring of L-dopa, rendering them highly soluble and stable to auto-oxidation when compared to L-dopa. Dopa phosphates are readily taken up by melanoma cells in culture and converted to L-dopa and inorganic phosphate by cellular phosphatases, making them useful for studying L-dopa effects in vivo. Here we investigated the effects of dopa phosphates on receptors for MSH in cultured melanoma cells. We found that dopa phosphates caused a 3-fold stimulation of MSH binding capacity by the cells which probably occurred through an increase in the number of receptors for MSH with no apparent change in affinity of the receptors. The increased binding capacity for MSH was followed by increased cellular tyrosinase activity and melanogenesis. Thus dopa phosphates and/or L-dopa can act as regulators of the MSH receptor system. The observations suggest a novel mechanism for regulation of hormonal responsiveness: hormonal signal amplification by a metabolite in the target pathway.     

Differences in sensitivity between human, mouse and Chinese hamster cells to killing by monochromatic ultraviolet light

Irradiation of human (IMR-91), mouse (10T1/2) and Chinese hamster (V79) fibroblasts with monochromatic ultraviolet light (u.v.) in the far-, mid-, and near-u.v. regions resulted in cell-strain-specific changes in sensitivity as a function of the wavelength used. The data suggested cell-strain-specific action spectra for cell killing by ultraviolet light that did not correlate with the ability of examined cells to excise pyrimidine dimers.     

MSH binding in bomirski amelanotic hamster melanoma cells is stimulated by L-tyrosine

Bomirski Ab amelanotic melanoma cells have recently been shown to undergo striking phenotypic changes when precursors of the melanogenic pathway, L-tyrosine and L-dopa, are added to the culture medium. The changes include increased tyrosinase activity andde novo synthesis of melanosomes and melanin. L-tyrosine and L-dopa appeared to elicit these responses through separate but overlapping regulatory pathways. Here we show an additional effect of L-tyrosine: stimulation of MSH binding capacity. Cells cultured for 24–48 hours in the presence of 200 M L-tyrosine display a 3–4 fold increase in their ability to bind¹²⁵l--MSH. L-dopa did not stimulate MSH binding under the same conditions. In control experiments neither L-tyrosine nor L-dopa had any effect on insulin binding. The amelanotic cells respond to MSH with increased dendrite formation, increased tyrosinase activity without melanin production, and decreased growth rate.     

Interactions between human immunodeficiency virus type 1 and human cytomegalovirus in human term syncytiotrophoblast cells coinfected with both viruses.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.     

Internal binding sites for MSH: analyses in wild-type and variant Cloudman melanoma cells

Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient (8-80%) and centrifuged (156,000g, 60 min); fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: 1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. 2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. 3) Most of the internal binding sites were not as dense as fully melanized melanosomes. 4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. 5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) 6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.     

Interactions between insulin and the cyclic AMP system of Cloudman S91 mouse melanoma cells

Insulin inhibits the proliferation of wild-type Cloudman S91 mouse melanoma cells. The effects, which are mediated through specific, high-affinity receptors for insulin, appear to involve interactions with the cAMP system. Our evidence is as follows: (1) Cloudman cells have a cAMP requirement for proliferation and pigmentation. Exposure of cells to insulin results in a lowering of intracellular cAMP levels and inhibition of both cell division and pigment formation. (2) The effects of insulin are reversed by agents which raise cAMP levels, or by the cAMP analogue dibutyryl cAMP. (3) A mutant cell line with a temperature-dependent requirement for cAMP is most sensitive to the growth inhibitory effects of insulin when its requirements for cAMP are maximal. (4) Mutants selected only for alterations in their response to Insulin frequently have concomitant alterations in their cAMP systems. (5) The melanotropin-responsive adenylate cyclase system is stimulated following prolonged exposure of cells in culture to insulin. Although we do not know the mechanism(s) for the interactions between the insulin and the cAMP system, our initial findings suggest that protein phosphorylation/dephosphorylation reactions are involved.     

In situ melanin assay for MSH using mouse B16 melanoma cells in culture

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with alpha-MSH, [Nle4, D-Phe7]-alpha-MSH, [3'-iodo-Tyr2]-alpha-MSH, adrenocorticotropin (ACTH)(1-24), and ACTH(1-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of alpha-MSH was 27 pM, i.e., about five- to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.     

Supermelanotic hybrids between mouse teratocarcinoma and mouse melanoma cells

Each of four kinds of teratocarcinoma cells, OTT6050P, PCC4, PSA1 and LT, derived from 129 or LT mouse strain, was fused with B16-CAPr melanoma cells derived from C57BL/6J by using Sendai virus. The resultant hybrids were morphologically melanotic melanoma cells which were larger and more heavily pigmented than the parental B16-CAPr melanoma cells. The chromosome analysis and GPI electrophoresis demonstrated that all hybrids were products of fusion between a single teratocarcinoma cell and a single melanoma cell. The pigmentation in the hybrids between a 129 teratocarcinoma cell and a melanoma cell was much stronger than that in hybrids between an LT teratocarcinoma cell and a melanoma cell. This phenomenon was consistent with the difference of coat color between 129 and LT mouse strain. From these results, it was suggested that the genes of teratocarcinoma cells involved in the pigmentation are activated in the hybrids with B16-CAPr melanoma cells.     

Cell-cycle analysis of insulin binding and internalization on mouse melanoma cells

Binding of 125I-labeled insulin to the surface receptors of Cloudman S- 91 mouse melanoma cells (CCL 53.1) was studied at various phases (M, G1, S, and G2) in the cell cycle. Insulin-binding activity was persistently present during the cell cycle but the highest activity was noted at the S-phase. The insulin once bound to the cell surface receptors at any phase of the cell cycle was internalized and degraded, presumably through a lysosomal pathway. Insulin-indexing activity of melanoma cells was not affected by melanocyte-stimulating hormone.     

L-Tyrosine stimulates induction of tyrosinase activity by MSH and reduces cooperative interactions between MSH receptors in hamster melanoma cells

L-tyrosine, a precurosr to melanin, has recently been shown to be a regulator of the melanogenic pathway in some cultured melanoma cell lines. In this paper we demonstrated that L-tyrosine, besides increasing binding capacity for MSH, decreased cooperativity between MSH receptors and increased the level of tyrosinase induction by MSH. Apparently, regulation of MSH receptor activity by L-tyrosine involves specific changes in the interactions between the receptors and modification of the cellular responsiveness to MSH.     

The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells.

Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O₂ oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from ¹⁴C/³H leucine studies suggest that MSH may cause increased $\text{de}$$\text{novo}$ synthesis of tyrosinase.     

Biological effects and binding properties of transforming growth factor-beta on human oral squamous cell carcinoma cells

The effects of transforming growth factor-beta (TGF-beta) on three human oral squamous cell carcinoma cell lines, HSC-2, HSC-3, and HSC-4, were investigated. Although these cell lines were equally sensitive to epidermal growth factor, responses to TGF-beta were variable. Dose-dependent inhibition of cell growth and [3H]thymidine incorporation of HSC-4 were observed by the addition of TGF-beta, whereas growth inhibitory effects on HSC-2 and HSC-3 were marginal. Moreover, treatment of HSC-4 with TGF-beta led to a more than 300-fold increase in fibronectin secretion into the medium. In contrast, TGF-beta did not increase the secretion of fibronectin on HSC-2 and HSC-3. Scatchard analysis of the binding of TGF-beta suggested that all squamous cell carcinoma cell lines have similar binding properties, with two classes of binding sites for TGF-beta. Affinity labeling of 125I-TGF-beta to cell surface receptors revealed the two major affinity crosslinked bands with Mr values of 65 kDa (type I) and 280 kDa (type III). A concomitant loss of 85 kDa band (type II) was observed in all squamous carcinoma cell lines examined. Although the proportions of type I and type III receptors were variable, the type I receptor, which is reported to be the main functional receptor in mediating the TGF-beta action, was commonly observed in these squamous cell carcinoma cell lines. These results indicate that the heterogeneity in response to TGF-beta between cell lines may be due to the difference in the signal transduction pathway of TGF-beta.     

Kinetics analysis of binding between melanoma cells and neutrophils

It has been determined previously that polymorphonuclear leukocytes, or PMNs, can facilitate melanoma cell extravasation through the endothelium under shear conditions. The interactions between melanoma cells and PMNs are mediated by the beta2-integrins expressed by PMNs and intercellular adhesion molecules (ICAM-1) expressed on melanoma cells. In this study, the kinetics of these interactions was studied using a parallel plate flow chamber. The dissociation rates were calculated under low force conditions for ICAM-1 interactions with both beta2-integrins, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), together and separately by using functional blocking antibodies on PMNs. The kinetics of PMNs stimulated with IL-8 was also determined. It was concluded that the small number of constitutively expressed active beta2-integrins on PMNs are sufficient to bind to ICAM-1 expressed on melanoma cells and that the intrinsic dissociation rate for these adhesion molecules appear to be more dependent on what method is used to determine them than on what cells express them.     

Interactions between diploid embryonal carcinoma cells and early embryonic cells

Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.     

Direct and indirect effects of ultraviolet light on the mutagenesis of parvovirus H-1 in human cells.

U.v. radiation is directly mutagenic for the single-stranded DNA parvovirus H-1 propagated in human cells. Mutation induction in the progeny of u.v.-irradiated virus increased linearly with the dose and could be ascribed neither to an increased number of rounds of viral replication nor to the indirect activation of an inducible cellular mutator activity by the u.v.-damaged virus. The level of mutagenesis among the descendants of both unirradiated and u.v.-damaged H-1 was enhanced if the host cells had been exposed to sublethal doses of u.v. light before infection. This indirect enhancement of viral mutagenesis in pre-irradiated cells was maximal at multiplicities lower than 0.2 infectious particles/cell. The frequency of mutations resulting from cell pre-irradiation was only slightly higher for u.v.-irradiated than for intact virus. Thus, the induced cellular mutator appeared to be mostly untargeted in the dose range given to the virus. U.v.-irradiation of the cells also enhanced the mutagenesis of u.v.-irradiated herpes simplex virus, a double-stranded DNA virus ( Lytle and Knott , 1982).