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J R Starkey

Publications and source records attributed to J R Starkey.

At least 37 records · Page 2Linked to original sources
PubMed1987

Comparison of basement membrane matrix degradation by purified proteases and by metastatic tumor cells.

We have examined the nature of biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) using different methodologies. The first strategy was quantitation of the release of surface-bound 125I and a second the documentation by SDS-PAGE of the appearance of putative cleavage products and the loss of high-molecular-weight components from the matrix. Basement membrane matrix bands resolved on SDS-PAGE were identified by their protease sensitivities as well as by Western immunoblots using monoclonal antibodies developed for this study. Radioiodinated components were found predominantly at positions on the gel equivalent to 160-200 kd and 400 kd proteins. Since these labeled moieties were sensitive to bacterial collagenase digestion and stained with anticollagen type IV antibodies, they were determined to represent various configurations of collagen type IV. Several other lower-molecular-weight bands also stained with the anticollagen IV antibodies. Monoclonal antibodies reactive with laminin exhibited a complex staining pattern on the gels, which included the expected 200 and 400 kd components. We confirmed that lens capsule basement membrane contained only a single heparan sulfate glycosaminoglycan species, and tumor cell-induced glycosaminoglycan degradation within the basement membrane matrix was detected using cellulose acetate electrophoresis. Distinctive putative cleavage products were resolved on SDS-PAGE gels from matrices subjected to digestion by a variety of purified proteases as well as by metastatic tumor cells or their conditioned media. Tumor cells of different histiotypes produced different characteristic cleavage patterns, suggestive of the existence of several pathways of matrix degradation. Overall, primary tumor cells exhibited a greater degradative activity towards the basement membrane matrix than did long-term tissue culture-passaged cells. The same tumor cell line could exhibit considerably different patterns of both protein and glycosaminoglycan degradation depending on recent culture history. The relevance of these biochemical studies to the pathogenesis of malignant neoplasms is shown by: 1) the evaluation of degradative activities of B16 tumor cell populations exhibiting enhanced lung-colonizing phenotypes, and 2) the ability of a known antimetastatic moiety with antiprotease activity (Haementeria leech species salivary gland extract) to protect matrix components from degradation by tumor cell-conditioned medium.

Animals↗
PubMed1987

The nature of tumor presentation in the animal changes the effects of levamisole treatment on metastasis.

We have investigated the ability of levamisole (LMS) to modulate growth and metastasis of a rat hepatocarcinoma. LMS treatment decreased spontaneous metastasis to the lungs and lymph nodes, while it increased tumor lung colonization following intravenous tumor cell inoculation. Both serum immunoglobulin (Ig) and circulating immune complex (CIC) levels were higher than normal in tumor-bearing rats. LMS treatment did not alter these parameters in the lung colony assay, while a small decrease of serum IgG was noted for LMS treated animals in the spontaneous metastasis assay. We found no convincing evidence for CIC levels, immunoglobulin isotype shifts or induced changes in natural killer (NK) cell reactivity being involved in the observed LMS modulation of tumor metastasis. The nature of the presentation of the tumor in the animal, however, appeared to be critical in determining the metastatic response to LMS therapy.

Animals↗
PubMed1987

Dietary restriction of tyrosine and phenylalanine: inhibition of metastasis of three rodent tumors.

The effects of a combined dietary restriction of tyrosine and phenylalanine on metastasis were investigated with the use of 3 rodent tumor cell lines: B16-bladder 6 (BL6) melanoma inoculated into (C57BL/6 X DBA/2)F1 mice, Lewis lung (3LL) carcinoma inoculated into C57BL/6 mice, and RT7-4bs hepatocarcinoma inoculated into BD-IV rats. When examined for effects on spontaneous metastasis, dietary restriction of tyrosine and phenylalanine had no effect on metastasis to draining lymph nodes in either BL6 or 3LL tumors. However, the restriction did reduce metastasis of RT7-4bs cells to draining lymph nodes by 60%. In all tumor systems, the dietary restriction effectively inhibited the subsequent growth of lymph node tumors. The most marked effect of the dietary restriction was on spontaneous hematogenous metastasis, which was almost totally blocked for BL6 cells and was reduced by 85% for RT7-4bs cells. Tumor-associated splenomegaly also was completely inhibited in 3LL tumor-bearing mice. The selective dietary amino acid restriction failed to reduce initial lung colonization in the experimental metastasis assay but clearly inhibited subsequent tumor outgrowth in the lungs. These findings demonstrate that modification of host nutritional status by restriction of the dietary intake of tyrosine and phenylalanine exerts a dramatic antimetastatic effect directed particularly on spontaneous hematogenous metastasis. Although the preliminary data suggest a primary modulating effect on tumor cell populations growing in diet-restricted animals to reduce inherent metastatic ability, the actual mechanisms remain to be defined.

Animals↗
PubMed1986

The role of tumor-cell surface carbohydrate in experimental metastasis.

Quantitative studies on the binding of concanavalin A (Con A) and wheat-germ agglutinin (WGA) to a series of rat hepatocarcinoma metastatic variants revealed a positive correlation between the amount of cell-surface-bound lectin and lung colonization potential. Scatchard analysis of Con A and WGA binding to 10 individual clones isolated from a subcutaneous (s.c.) tumor transplant and to tumor-cell isolates from 10 individual spontaneous lung metastases from the same animal showed diverse binding characteristics for these cell populations. Nevertheless, the expression of Con A receptor sites accurately predicted the lung colonization potential of 3 isolates from the lung metastases. Higher lectin binding curves were observed for the clones from the subcutaneous tumor than for the isolates from lung metastases. These data suggest that a high Con-A binding potential is indicative of a high lung colonization potential for these hepatocarcinoma cells, but that this phenotype may be rapidly lost during tumor outgrowth in the lungs. The binding of tumor cells to vascular endothelial cell monolayers was inhibited in the presence of Con A; however, no inhibition was observed with 2 other lectins. Attachment of tumor cells to endothelial cell monolayers was also inhibited by the monosaccharides methyl alpha-D-mannopyranoside and N-acetyl-D-galactosamine. Other monosaccharides tested did not alter the attachment of tumor cells to endothelial cell monolayers.

Animals↗
PubMed1986

Interaction between specific dietary factors and experimental chemotherapy of metastatic melanoma.

The single and combined effects of (a) dietary restriction of phenylalanine and tyrosine, (b) levodopa methylester chemotherapy, and (c) megadose sodium ascorbate supplementation on experimental metastasis was determined in B16-BL6 melanoma. Dietary restriction and levodopa methylester therapy inhibited tumor outgrowth, whereas ascorbate alone was inactive. In combination, however, the effect of dietary restriction and levodopa methylester chemotherapy was augmented by sodium ascorbate. Tumor cells surviving this combination therapy (treated population) were isolated from the lungs of treated mice, and proved to be tumorigenic when inoculated SC into the back of naive mice. The resulting tumors grew more slowly than those produced by inoculation of similarly isolated control cells (control population), irrespective of whether the diet was adequate or deficient in phenylalanine and tyrosine. Failure of the treated tumor cell population to exhibit reduced sensitivity to the combination chemotherapy or, unlike the control population, to exhibit variation in pigmentation levels, suggests that the restriction of phenylalanine and tyrosine during drug therapy alters the tumor response to reduce heterogeneity and perhaps interferes with the emergence of drug resistance.

Animals↗
PubMed1985

Cell surface thiols, but not intracellular glutathione, are essential for cytolysis by a cloned murine natural killer cell line.

Cell surface thiols are required for a line of cloned murine natural killer lymphocytes to bind to and lyse tumor target cells. These lymphocytes neither bound to nor killed YAC-1 or G1Tc cells when the effector lymphocyte cell surface thiols were covalently coupled with the non-penetrating reagent, monobromotrimethylammoniobimane (qBBr). A limited number of thiol-bearing proteins were identified by gel electrophoresis on the cell surface using the fluorescence of the group that remains associated with the sulfur molecule. These results indicate that either one or more of these reactive proteins or different cell surface thiol-bearing molecules present at low frequencies are crucial to lymphocyte binding and killing. In contrast, we found little evidence that intracellular thiols are required for natural killer cell activity. Killing was relatively unimpaired when over 90% of lymphocyte glutathione was depleted with DL buthionine-S,R-Sulfoximine (BSO). Blocking the intracellular or the extracellular thiols of tumor targets had no effect on their ability to be lysed. Based on these data, we suggest that infrequently expressed extracellular thiols are required either for the conformation or for the disulfide crosslinking of proteins that participate in lymphocyte-mediated cytolysis.

Animals↗
PubMed1984

Influence of migratory blood cells on the attachment of tumor cells to vascular endothelium.

The purpose of this study was to determine whether the presence of migratory blood cells in association with tumor emboli had the capacity to alter the attachment of tumor cells to vascular endothelium. Highly metastatic RT7-4bs rat hepatocarcinoma cells were labelled with [125I]UdR before being allowed to form mixed cellular spheroids incorporating resident peritoneal macrophages, activated peritoneal macrophages, polymorphonuclear leukocytes, splenic T lymphocytes, or splenic B lymphocytes derived from both normal and tumor-bearing animals. The presence of polymorphonuclear leukocytes or activated macrophages led to a considerable increase in the number of tumor cells attaching to endothelial cell monolayers in vitro. The presence of T or B lymphocytes from either normal or tumor-bearing rats was without effect on tumor attachment to endothelium. Increased tumor cell retention in the lungs was evident for mixed spheroids containing tumor cells and polymorphonuclear leukocytes compared to homotypic tumor spheroids composed of tumor cells alone. Furthermore, preinjection of polymorphonuclear leukocytes intravascularly or inoculation of tumor cells as heterotypic spheroids containing polymorphonuclear leukocytes increased lung colony formation over that obtained after inoculation of tumor cells alone. Several simple sugars were tested for their ability to block tumor cell, polymorphonuclear leukocyte or activated macrophage binding to endothelium in vitro. The results indicate that the glycosylated cell surface components mediating tumor cell attachment to endothelium are not identical with those mediating attachment of either polymorphonuclear leukocytes or activated macrophages. Medium conditioned during mixed spheroid formation was without effect on tumor cell attachment to endothelium. We conclude that the presence of some, but not all classes of leukocytes can modulate tumor cell attachment to vascular endothelium, an effect most likely mediated by a mechanism involving direct contact between the leukocytes and the endothelial cell monolayer.

Animals↗
PubMed1984

Immunologic enhancement of experimental metastasis in the rat.

Using a series of immunologically cross-reactive metastatic tumor variants, we demonstrate that serum from animals bearing pulmonary tumor colonies possesses enhancing properties in the experimental metastasis (lung colony) assay. Enhancement is produced by chronic serum administration and promotes the growth of tumor cells arrested in the lungs which would not otherwise proliferate to form grossly detectable lung nodules. Tumor-bearer serum from animals with lung colonies derived from the most highly metastatic variant examined is shown to possess enhancing properties in both BD-IX(H-1d) and BD-IV(H-1d) rat strains, while tumor-bearer serum from animals with lung colonies derived from the less metastatic parent tumor cell line possesses enhancing properties in the BD-IX rat strain only. Removal of immunoglobulin from enhancing serum by affinity column chromatography simultaneously removes the enhancing factor(s), and enhancing activity correlates with the presence of increased levels of Clq-binding immune complexes in the serum. Serum levels of immune complexes are shown to be more elevated in serum from animals bearing lung colonies derived from the most highly metastatic variant. The enhancing moieties are shown to bind to concanavalin A, but not to staphylococcal protein A, and the active fraction elutes from concanavalin A-Sepharose with alpha-methyl-mannoside. Consideration of immunoprecipitation studies on whole and fractionated enhancing sera, along with studies on affinity purified isotype fractions reveals that the activity resides with antibodies of IgG2b subclass.

Animals↗
PubMed1984

Interaction of metastatic tumor cells with bovine lens capsule basement membrane.

The use of bovine lens capsule basement membrane as a model substratum for studies of invasion and extravasation by metastatic tumor cells is described. The abilities of three independently isolated pairs of metastatic variant cell lines to digest the purified substrates, laminin, type IV collagen, and type I collagen, were compared with their abilities to solubilize isotope from 125I-labeled lens capsule basement membrane matrix. The cell lines used were +SA and -SA mouse mammary adenocarcinoma cells, RT7-4bs and RT7-4b-Ls rat hepatocarcinoma cells, and B16-F1 and B16-F10 mouse melanoma cells. In general, imperfect correlations of lytic activity with metastatic ability were found for the purified substrate digestions, but, for each pair of variants, the more metastatic tumor cell line was always able to solubilize more surface-bound isotope from the lens capsule. Visual evidence of tumor cell-associated digestion of lens capsule basement membrane was obtained using transmission electron microscopy. Mouse mammary carcinoma cells attached more rapidly to lens capsule than to endothelial cell monolayers or tissue culture plastic. We next added endothelial cells to the model substrate. Aortic endothelial cells grew well on lens capsules without apparent synthesis of additional basement membrane matrix. In additional studies, the lens capsule was used in a chamber apparatus to demonstrate that cellular invasion of the full thickness of this basement membrane structure could be demonstrated and readily quantitated. Our results indicate that bovine lens capsule is a particularly versatile basement membrane structure useful for studies of tumor cell invasion and extravasation. In addition, the comparison of purified substrate digestions with lens capsule matrix digestion indicates the desirability of also using a matrix digest when correlating lytic abilities of tumor cells with their metastatic abilities.

Adenocarcinoma↗
PubMed1983

Natural killer cell activity in fawn-hooded rats.

Fawn-hooded (FH) rats were shown to lack the genetically conditioned defect of natural killer (NK) activity hypothesized to be present by analogy with the Chediak-Higashi syndrome (CHS) in mice and human beings. In 4-h 51Cr release assays, splenic NK cells from FH rats killed YAC-1, RL male l and G1-TC tumor targets without deficiency based upon comparison with cells from BD-IV, BD-IX and NBR inbred rat strains. Progeny of BD X FH F1 rats backcrossed to FH failed to reveal a correlation of reduced NK activity and dilute coat color. From these, and other data presented, it is concluded that despite similarities in coat color dilution and platelet storage pool deficiency. FH rats do not closely resemble CHS mice or human beings in having deficient NK activity and cannot be considered the rat homolog of the CHS.

Animals↗
PubMed1983

Alcohol and related dietary effects on mouse natural killer-cell activity.

Natural killer (NK) cell activity of spleen cells from female C57BL/6 mice receiving 10% w/v alcohol solution for 4 weeks was studied in mice fed a nutritionally complete crystalline amino-acid diet and in mice fed diets moderately deficient in (i) tyrosine and phenylalanine or (ii) methionine. Natural killer cell activity was determined in a 4-hr cytolytic chromium-release assay against YAC-1 lymphoma cells. Alcohol consumption did not effect NK cell-mediated lysis irrespective of nutritional status; however, NK-cell activity was depressed in mice fed the tyrosine- and phenylalanine-deficient diet and was enhanced in mice fed the methionine-deficient diet. These data suggest that the changes in immune function often observed in alcoholics may be more closely linked to dietary and nutritional status than to the direct effects of the ingested alcohol.

Alcoholism↗
PubMed1983

In vitro effects of protease inhibitors on murine natural killer cell activity.

To test whether proteolytic events are involved in natural killer (NK) cell mediated lysis of tumour cells, twenty-three different protease inhibitors were added to in vitro assays of natural killer cell reactivity. Of all of the materials tested, only tosyl-L-lysine chloromethyl ketone (TLCK), tosyl-L-phenylalanine chloromethyl ketone (TPCK) and benzamidine unequivocally inhibited killing at concentrations approaching those needed to affect appropriate purified proteases. All of the effective inhibitors, and none of the others tested, inhibited binding of effector to target cells. The action of TLCK was focused on both effector and target cells, in that cytolysis was completely inhibited by a 1 hr pretreatment of effectors with 10(-4) M TLCK, and 60% inhibited by a 1 hr treatment of targets only.

Animals↗
PubMed1982

Immunoselection of tumor variants resistant to antibody-mediated cytotoxicity. Their immunologic and metastatic characterization.

The immunological characteristics of two series of metastatic variants of restricted genetic origin were related to their lung-colony-forming potential. A series of metastatic variants was isolated from a tumor-cell population in which heterogeneity appeared following short-term in vivo passage, while a second series of variants were immunoselected in vitro for resistance to antibody-complement-mediated cell lysis. In the case of the first series, the sensitivity of the individual variants to cell-mediated and antibody-complement-mediated cytotoxicity appeared to be partly determined by the number and rate of loss of antibody-binding sites on the cell surface. These characteristics also correlated with the efficiency of experimental metastasis in the animal. We compared the results reported herein with our previous studies of nonimmune parameters for this series of metastatic variants, and we conclude that immunological differences can be important to the efficiency of lung-colony formation. However, in the case of the second series of variants, despite selection in vitro for resistance to antibody-complement-mediated cell lysis, the behavior of these variants in the lung colony assay could not be predicted by the immunologic parameters examined.

Animals↗
PubMed1981

In vitro characteristics of metastatic variant subclones of restricted genetic origin.

We have studied several metastatic variant cell lines derived from a common clonal origin and their transformed and untransformed parental cell lines. A number of in vitro characteristics were examined for each tumor line and these properties were correlated with the ability of the tumor cells to form pulmonary nodules in an experimental metastasis assay. Direct correlations with metastatic behavior in the lung colony assay were found to exist with the amount of cell-bound Concanavalin A and the procoagulant activities of cell lysates. In vitro parameters that did not correlate with the metastatic phenotype were: population doubling times in culture, saturation density achieved in culture, the number of colony-forming cells shed from confluent cultures, rates of cellular attachment to homotypic or heterotypic cell monolayers, plasminogen-activator production and procoagulant activity produced in serum-free conditioned medium.

Animals↗
PubMed1980

Role of NK cells in tumour growth and metastasis in beige mice.

Although natural killer (NK) cells are thought to give the host a spontaneous resistance against tumours and have been postulated to act in vivo as surveillor cells, definitive data in support of these hypotheses has not been obtained. Recently the beige (bg) mouse, a morphological homologue of the human Chediak-Higashi (CH) syndrome, was shown to be deficient in NK activity. Specifically, spleen cells of bg mice were demonstrated to be incapable of in vitro natural cytotoxicity against tumour cells. We report here that a tumour line, modified to be sensitive to NK cytotoxicity by in vitro culture, demonstrated in vivo an increased growth rate, faster induction time and an increased metastatic capability in bg compared to control mice. This was not found with a tumour line insensitive to NK activity (without in vitro culture). In vivo activation of NK cells in bg and control mice resulted in a decrease in tumour growth rate and metastatic frequency. These results demonstrate that NK cells have an important function in the host's control of tumour growth and metastasis.

Animals↗
PubMed1980

Role of natural killer cells in tumor growth and metastasis: C57BL/6 normal and beige mice.

The role of natural killer (NK) cells in tumor growth and metastasis was studied in syngeneic normal and beige inbred C57BL/6 mice. Mice with the beige point mutation have been shown to be deficient in nonstimulated NK activity. Tumor-passaged B16 malignant melanoma cells were refractory to NK activity as determined by in vitro assay, but after in vitro culture they became sensitive to NK activity. The NK-insensitive B16 tumor grew and metastasized similarly in normal and beige mice. However, the NK-sensitive B16 tumors grew more slowly and produced fewer metastases in normal mice than in NK-deficient beige mice. Activation of NK cells by lymphocytic choriomeningitis virus infection decreased the rate of growth and number of metastases of both NK-sensitive and NK-insensitive tumors in both normal and beige mice. These results suggest the importance of NK cells as a determinant of tumor growth and metastasis.

Animals↗