Biomedical subjects

A F Chambers

Publications and source records attributed to A F Chambers.

At least 109 records · Page 6Linked to original sources

PubMed1992

Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells.

We previously found that the T24 Ha-ras oncogene induces metastatic ability in NIH 3T3 cells and that this change depends on expression of the ras oncogene. As part of our studies on mechanisms by which ras may induce metastasis, we investigated expression and activity of two cysteine proteinases, cathepsin L (major excreted protein) and cathepsin B, as well as cysteine proteinase inhibitor activity, in ras-transformed NIH 3T3 cells. In a series of cel lines that expressed differing amounts of ras, we found a good correlation between levels of ras expression and cathepsin L expression (r = 0.80). There was also a good correlation between secreted procathepsin L protein levels and experimental metastatic ability (r = 0.88). We found a similar but less strong association between cathepsin B levels and metastatic ability in these cells (r = 0.76 and r = 0.72 for 2.2-kb and 4.1-kb transcripts, respectively). Functional cathepsin L plus B activity (both secreted and cell-associated) was found to be higher in ras-transformed cells and was dependent on cell confluency in culture. Coupled with increased expression and activity of cysteine proteinases, ras-transformed NIH 3T3 cells showed reduced cysteine proteinase inhibitor activity. We conclude that the balance between expression of cysteine proteinases and their inhibitors may be coregulated by ras expression. Our results suggest that ras-induced increases in production of degradative enzymes such as cathepsins L and B, along with decreased activities of their inhibitors, may contribute to the increased malignant properties of ras-transformed NIH 3T3 cells.

3T3 Cells↗

PubMed1992

Up-regulation of TIMP-1 expression in B16-F10 melanoma cells suppresses their metastatic ability in chick embryo.

Clonal B16-F10 cell lines with increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) have been generated by transfection with a TIMP-1-containing expression vector. The parental B16-F10 and control 1-2 cells, and two TIMP-1 up-regulated clones (2-10, 6-5), were studied for their growth characteristics in tissue culture and their experimental metastatic ability in the chick embryo. Both of the TIMP-1 up-regulated clones showed slower in vitro growth and had lower saturation densities. Both clones were also less metastatic following intravenous injection into chick embryos, and formed significantly fewer metastatic tumors in the chorioallantoic membrane and in the liver than did parental B16-F10 and control cells. Furthermore, the size of tumors formed by TIMP-1 up-regulated cells was significantly reduced in comparison to the tumors produced by B16-F10 or control cells. Our results show that malignant cell lines genetically modified to express increased levels of TIMP-1 exhibit a suppressed experimental metastatic ability in vivo. We propose that TIMP-1 suppresses metastatic ability by decreasing both invasive and growth abilities.

Animals↗

PubMed1992

Intravital videomicroscopy of the chorioallantoic microcirculation: a model system for studying metastasis.

The chick embryo is a useful model for studying hematogenous metastasis. Cancer cells injected into veins of the chorioallantoic membrane (CAM) circulate briefly through all tissues but form metastases predominantly in the CAM. This respiratory organ is particularly suitable for intravital microscope because of its accessibility without the need for surgery and the density and planar configuration of its vessels (which we confirmed by microcorrosion casting). Using an inverted microscope with oblique transillumination for high-resolution images and epifluorescence to identify labeled B16F1 melanoma cells, we studied successive stages of metastasis formation in the CAM in vivo. By 2 min postinjection (pi) all cancer cells had become arrested within the microvasculature. This initial arrest appeared to be due to size restriction, based on measurements of cell and vessel diameters. At 15-60 min pi, trapped cells were seen in tapering arterioles (27%), orifices from arterioles to the capillary plexus (61%), or in the plexus itself (12%). Some cells had extravasated into the underlying mesenchyme by 3 hr (pi), and at 24 hr all cancer cells had completed this process. The mean rate of migration out of capillary lumens was approximately 1 micron/hr. Micrometastases grew in a planar configuration just beneath the capillary plexus, with a cell doubling time of approximately 24 hr. Our technique is also applicable to other tumor types and host animals and provides a powerful tool to complement studies on the molecular basis of metastasis.

Allantois↗

PubMed1992

Mechanisms of oncogene-mediated alterations in metastatic ability.

Transfected ras oncogenes have been shown to induce metastatic properties in some cells. This altered behavior is likely due to changes in ras-mediated signal transduction pathways, resulting in altered expression of genes important to metastasis. Clarification of the mechanisms by which ras is able to induce metastatic ability in model systems will improve our understanding of tumor progression, even in those cells in which ras activation has not been implicated. Many of the consequences of ras expression also have been detected in cells that have become metastatic in the absence of altered ras, suggesting that there is a set of common changes that can lead to metastasis, with multiple signals capable of eliciting these changes. We have identified several changes in metastatic, ras-transformed NIH 3T3 cells that may contribute to their increased malignancy, including expression of proteolytic enzymes and their inhibitors, and adhesive and calcium-binding proteins. Not all cells, however, respond in this way to expression of oncogenic ras. We have found that murine LTA cells, which are tumorigenic but nonmetastatic, are ras resistant and remain nonmetastatic when expressing high levels of transfected ras, in contrast to NIH 3T3 cells, which are ras sensitive and become both tumorigenic and metastatic in response to comparable levels of ras. LTA cells differ in their patterns of gene expression in response to ras when compared with NIH 3T3 cells, suggesting that the two cell lines process the ras signal differently. Here we review our results with ras-transfected NIH 3T3 and LTA cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Different patterns of gene expression in ras-resistant and ras-sensitive cells.

We have shown previously that nontumorigenic NIH 3T3 cells can be made tumorigenic and metastatic by transfection and expression of activated ras, whereas in LTA cells, which are tumorigenic but nonmetastatic, the degree of malignancy is not altered by ras. To investigate possible mechanisms of natural ras resistance, we compared the expression patterns of several genes thought to be involved in ras-induced metastatic progression in LTA (ras-resistant) and NIH 3T3 (ras-sensitive) cells, before and after constitutive expression of transfected T24-H-ras. We examined the expression of the nuclear "early-response" genes jun and fos and the "tumor-suppressor" retinoblastoma (Rb) gene, as well as genes involved in invasion (major excreted protein [MEP], tissue inhibitor of metalloproteinases [TIMP]), and cell adhesion (secreted phosphoprotein 1 [SPP1; also known as osteopontin]). We found distinct differences in both the basal and ras-induced levels of expression of most of these genes in LTA versus NIH 3T3 cells. High levels of MEP and low levels of TIMP were induced in ras-transfected NIH 3T3 cells, whereas LTA cells showed intermediate levels of MEP and high levels of TIMP that were only marginally affected by the expression of transfected ras. Similarly, SPP1 expression was strongly induced by ras in NIH 3T3 cells but was repressed by ras in LTA cells. Enzymogram assays for functional gelatinase activity showed an increase in 67-kd and 62-kd bands in NIH 3T3 cells in the presence of ras. LTA cells showed no gelatinolytic activity in the presence or absence of ras. Data from an in vitro assay for chemoinvasiveness showed a pattern as predicted from the expression of invasion-related genes; chemoinvasiveness in ras-transfected NIH 3T3 was greater than in LTA and ras-transfected LTA cells, which was greater than in NIH 3T3 cells. Differences in expression of the genes examined are believed to contribute to the ras responsiveness of NIH 3T3 cells and the ras resistance of LTA cells.

Animals↗

PubMed1991

Malignant properties of P635 glioma are independent of glial fibrillary acidic protein expression.

To examine the relationship between the malignant behavior of rat glioma cells and expression of the differentiation antigen glial fibrillary acidic protein (GF), we assayed the tumorigenicity and metastatic ability of P635 and its GF+ or GF- clones. We injected P635 (GF+) and clone 45 (GF-) cells intramuscularly in nude mice. Both lines formed local tumors which metastasized to lungs with equal efficiency. We then injected these lines intravenously in nude mice. Both produced experimental metastases in lungs and other organs with equal efficiency. Finally we injected P635 and several GF+ and GF- clones into the chorioallantoic membrane veins of naturally immune-deficient chick embryos and measured cell growth in embryonic liver. We again found that all clones survived and grew equally well. P635 cells are capable of extracranial growth and metastasis, two important features of the malignant phenotype. We conclude that GF in P635 is a neutral marker with respect to tumorigenicity and metastatic ability.

Animals↗

PubMed1991

Gene expression and metastasis of somatic cell hybrids between murine fibroblast cell lines of different malignant potential.

We have used somatic cell hybrids to study the relationship between ras sensitivity, metastasis, and the expression of ras-responsive or "metastasis-associated" genes. We have previously shown that NIH 3T3 cells are nontumorigenic, but are made metastatic by transfection and expression of activated ras (i.e., they are ras-sensitive). LTA cells, however, are initially tumorigenic, but nonmetastatic, and are not altered in malignancy by ras (i.e., they are ras-resistant). We also have shown that patterns of expression of ras-responsive and "metastasis-associated" genes differ markedly between these two cell types. In the present work, we have constructed three sets of somatic cell hybrids: NIH 3T3 X LTA cells (designated NL), NIH 3T3 X ras-transfected LTA cells (designated NLR), and LTA X ras-transfected NIH 3T3 cells (designated LNR). In all three sets of cell hybrids, pooled clones were found to be highly metastatic in the chick embryo assay, suggesting complementation had occurred. Those cell hybrids that contained ras (NLR and LNR hybrids) were significantly more metastatic than those that did not (NL hybrids). Selected clones of low and high metastatic ability from both NL and LNR fusions were examined for tumorigenicity and "experimental" metastatic ability in nude mice, as well as for expression of several genes thought to be involved in ras-induced progression and malignancy. Patterns of expression of these genes showed a relationship to level of malignancy of the hybrids and demonstrated a responsiveness to the expression of activated ras. These results suggest that the complementation of phenotype observed in the hybrids may arise through a gene regulatory factor(s) supplied by the NIH 3T3- to the LTA-derived parent.

Animals↗

PubMed1990

Secreted phosphoprotein mRNA is induced during multi-stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts.

Secreted phosphoprotein I (SPP), also known as 2ar, osteopontin, 44-kDa bone phosphoprotein, bone sialoprotein I, and transformation-related phosphoprotein, is a 41.5-kDa glycosylated phosphoprotein secreted by many mammalian cell lines and expressed in a limited set of tissues. Using a cDNA probe, we found that SPP mRNA, which is barely detectable in normal mouse epidermis, was expressed at moderate-to-high levels in 2 of 3 epidermal papillomas and at consistently high levels in 7 of 7 squamous-cell carcinomas induced by an initiation-promotion regimen. This contrasts with the transient induction we had previously observed after a single application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In a set of 5 independently isolated T24-H-ras-transfected mouse C3H 10T1/2 cell lines, the levels of SPP mRNA correlated well with ras mRNA levels and with both experimental and spontaneous metastatic ability. SPP mRNA expression was also elevated in a derivative of mouse LTA cells transfected with genomic DNA from B16F1 melanoma cells and selected for increased experimental metastatic ability in the chick embryo. This apparent association of SPP expression with invasion, progression and metastasis, along with the presence of a functional ArgGlyAsp (RGD) cell adhesion site in SPP (osteopontin), leads us to propose that SPP may act as an autocrine adhesion factor for tumor cells.

Animals↗

PubMed1990

Some ras-transformed cells have increased radiosensitivity and decreased repair of sublethal radiation damage.

We examined the effect of 60Co irradiation on the clonogenic survival of rat NRK cells, NRK cells carrying a temperature-sensitive viral K-ras oncogene (tsK-NRK), mouse NIH 3T3 cells, and NIH 3T3 cells transformed with the human bladder cancer (T24) H-ras oncogene (PAP2). We tested the hypothesis that ras oncogene expression renders cells more resistant to radiation, but found in both systems that ras-transformed cells were more, not less, sensitive to radiation. We also found indications of altered repair of sublethal radiation damage. PAP2 cells were more sensitive to radiation than NIH 3T3 cells. Increased sensitivity was reflected in a decreased shoulder region of the survival curve with little effect on its slope (D0). TsK-NRK cells were also slightly more sensitive to radiation than NRK and exhibited decreased repair of sublethal damage at both the permissive and nonpermissive temperatures. Thus, we found that expression of ras oncogenes is not always associated with increased radiation resistance. In summary, our results suggest that (1) ras oncogene expression in some cells may be associated with increased, rather than decreased, radiation sensitivity, and (2) ras oncogene expression may alter the shoulder region of the dose response curve, suggesting changes in the repair of sublethal radiation damage.

Animals↗

PubMed1990

Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin: tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells.

A mouse mRNA, provisionally designated 5B10, has been cloned based on its inducibility by serum in quiescent murine fibroblasts. Here we report the full-length complementary DNA sequence and a partial characterization. There are about five copies of the gene in the mouse genome. Sequence analysis of the 5B10 coding region reveals 94 and 97% amino acid identity to human and rat calcyclin, respectively. Although the coding region has been highly conserved during evolution of the rodent and human genomes, the untranslated flanking sequences differ significantly. A protein of Mr about 8000 was produced by in vitro translation of the mRNA transcribed in vitro from 5B10 complementary DNA in a riboprobe vector. An antiserum raised against a portion of the predicted human calcyclin protein cross-reacted with this mouse protein. 5B10 mRNA was found in greatest amount in organs containing proliferating cells, e.g., epidermis, skin, stomach, uterus of pregnant mouse, placenta, and decidua. Brain, liver, mature thymus, and skeletal muscle had little or no detectable 5B10 mRNA. 5B10 mRNA levels were higher in cells treated with 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate than in their normal counterparts, suggesting a role in tumorigenesis. In addition, high 5B10 mRNA levels were associated with metastatic ability in a series of ras-transformed cells, in proportion to levels of ras p21 expressed by the cells, implicating 5B10 even more deeply in carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1990

ras-transformed NIH 3T3 cell lines, selected for metastatic ability in chick embryos, have increased proportions of p21-expressing cells and are metastatic in nude mice.

We previously reported that ras-transformed NIH 3T3 cells could be selected in vivo for increased metastatic ability after intravenous injection into chick embryos, and that the metastatic cell populations expressed increased levels of ras p21 protein. We have tested the metastatic ability of a series of these cells in nude mice, to determine if their properties in the chick embryo experimental metastasis assay predict their metastatic behavior in nude mice. We report here that cells selected for metastatic ability in the chick embryo are also metastatic when assayed in nude mice. We also asked whether the selected cells uniformly expressed higher levels of p21, using an immunocytochemical procedure. We found that p21 expression among individual cells in these populations was quite heterogeneous. There was a direct relationship between the proportions of p21-expressing cells and metastatic ability in both assays, with increased proportions of p21-expressing cells in cell lines selected for metastatic ability. Our results suggest that (a) the experimental (i.v.) metastasis assay in the chick embryo offers an efficient and cost-effective procedure for the identification and selection of cells that are also experimentally metastatic in nude mice, and (b) the metastatic properties of ras-transformed NIH 3T3 cells are due to individual cells that express increased amounts of p21.

Animals↗

PubMed1989

Psychiatric severity of illness. A case mix study.

This study was undertaken to determine if a measure of severity of illness for psychiatric patients, the Psychiatric Severity of Illness Index, could produce psychiatric case mix groups that are more homogeneous with respect to resource use than the diagnosis-related groups (DRGs). Psychiatric Severity of Illness data were collected on 1,672 cases in ten hospitals of various types. Of these cases, 1,418 had enough information in the medical record to be scored using the Psychiatric Severity Index, 1,173 of which were in MDC 19 (mental diseases and disorders). We found that four Psychiatric Severity of Illness groups explained between 34% and 50% of the variation in length of stay of the combined hospital data in MDC 19, whereas nine DRGs explained between 6% and 14%. DRGs subdivided by Psychiatric Severity of Illness groups explained between 40% and 54% of the variation in length of stay. The implications of these results for cross-hospital comparisons are discussed.

Contraceptives, Oral, Combined↗

PubMed1988

Identification of the major phosphoprotein secreted by many rodent cell lines as 2ar/osteopontin: enhanced expression in H-ras-transformed 3T3 cells.

/ar, a tumor promoter-inducible protein secreted by mouse JB6 epidermal cells, is the murine homolog of rat osteopontin, or 44 kD bone phosphoprotein. We report here that 2ar is also related to pp69, a major phosphoprotein secreted by normal rat kidney cells. Antisera raised against pp69 and against beta-galactosidase-2ar fusion proteins are able to immunoprecipitate the same major phosphoproteins, of apparent Mr 55-69 kD, secreted by several rat and mouse cell lines. The levels of secreted protein and cytoplasmic mRNA are dramatically elevated in NIH 3T3 cells transformed with the human bladder cancer T24 (H-ras) oncogene. These results and the work of Senger and colleagues (Cancer Res., 45, 5818-5823, 1985) imply that enhanced secretion of 2ar/pp69/osteopontin by transformation of a wide variety of mammalian fibroblasts and epithelial cells is often correlated with tumorigenicity.

Animals↗

PubMed1988

Clonal heterogeneity, experimental metastatic ability, and p21 expression in H-ras-transformed NIH 3T3 cells.

We examined individual clones of murine NIH 3T3 cells, transformed with the human bladder cancer (T24) H-ras oncogene, for p21 expression and for experimental metastatic ability in the immunodeficient chick embryo. We found that the clones were heterogeneous for both of these properties. In general, p21 expression was a good predictor of metastatic ability of the clones. Cells from poorly metastatic clones were passaged in the chick embryo metastasis assay to determine whether cells with increased metastatic ability could be selected. We found that the selected cells were more metastatic and that substantial increases in expression of p21 also accompanied this increase in metastatic ability. The relationship between p21 expression and metastatic ability appeared linear, with a high correlation coefficient (r = .85), suggesting that in this model system quantitative increases in metastatic properties can result from increased expression of the ras oncogene protein product p21.

Animals↗

PubMed1988

Rates of generation of methotrexate-resistant variants in cells temperature-sensitive for malignant transformation.

We used tsLA23-NRK cells, in which malignant transformation is under the control of a temperature-sensitive (ts) src oncogene, to examine the relationship between genetic instability and malignant transformation. Cells were maintained in vitro as either transformed cells (at 36 degrees C) or as normal cells (at 39 degrees C). Genetic instability was assessed in these two cell populations by determining (1) the frequencies of methotrexate (MTX)-resistant variants present in these populations, and (2) the rates of generation of MTX-resistant variants, as determined from Luria-Delbrück fluctuation analyses. We observed no significant differences, in either of these parameters of genetic instability, that could be attributed to transformation status. We conclude that in this defined cellular system there is no obligatory relationship between malignant transformation and increased genetic instability, as assessed by either frequencies or rates of generation of variants resistant to MTX.

Animals↗

PubMed1987

Dynamic heterogeneity: characterization of two cell lines derived from experimental lung metastases of mouse KHT fibrosarcoma.

We have recently demonstrated that KHT fibrosarcoma variant cells that form experimental metastases are generated by a stochastic process at high effective rates (approximately 10(-5)/cell/generation). The metastatic variant cells are unstable and are rapidly lost with an effective rate of reversion of approximately 10(-1)/cell/generation. In this study, we have examined the dynamics of generation of metastatic variant cells for two lines of KHT cells derived from individual experimental lung metastases in a single step selection. When grown in vitro, the KHT35-L1 cell population maintained a highly metastatic phenotype whereas the KHT3-L1c cell population reverted to a poorly metastatic phenotype. Our results indicate that there is an approximately 10-fold higher effective rate of generation of metastatic variant cells for KHT35-L1 cells relative to KHT3-L1c cells. Thus, a higher effective rate of generation of metastatic variants accompanies a stably expressed metastatic ability in fibrosarcoma cells and, as we have previously reported, in melanoma cells.

Animals↗

PubMed1986

Severity of illness within DRGs. Homogeneity study.

The authors assess the ability of the Severity of Illness Index to explain variability of resource use within each DRG. The data came from 15 hospitals, all of which had a HCFA DRG case mix index greater than 1. The data set comprised approximately 106,000 discharges, for which discharge abstract data, financial data, and Severity of Illness data were available. To pool the data over the 15 hospitals, the authors converted all charges to costs and normalized them to fiscal year 1983. Adjustments were also made for medical education and wage levels. The Severity of Illness Index explained more than 10% of the variability in resource use in 94% of the DRGs, which contained 97% of the patients, and more than 50% of the variability in resource use in 36% of the DRGs, which contained 24% of the patients. For the whole data set, DRGs explained 28% of the variability in resource use, and severity-adjusted DRGs explained 61% of the variability in resource use. Thus the Severity of Illness Index explained a large amount of the variability in resource use within individual DRGs as well as in the whole data set. This explanatory power remained when outliers were removed. These results go beyond previous studies that were based on six disease conditions and/or were analyzed only within individual hospitals. The findings indicate that the phenomenon of severity of illness differences within DRGs, and the corresponding differences in resource use, is consistent across 15 hospitals that represent all sections of the United States and all teaching types.

Costs and Cost Analysis↗

PubMed1985

Interhospital differences in severity of illness. Problems for prospective payment based on diagnosis-related groups (DRGs).

We evaluated the ability of the diagnosis-related-group (DRG) classification system to account adequately for severity of illness and, by implication, for the costs of medical care. Hospital inpatients on medicine, surgery, obstetrics/gynecology, and pediatrics services in six hospitals were evaluated to provide a spectrum of patient and hospital characteristics. This evaluation was based on data from a generic index of severity of illness obtained by trained personnel from a review of hospital charts after patient discharge. Within each DRG, substantial differences were found in the distribution of severity of illness in different hospitals. Some hospitals treated larger proportions of severely ill patients and had a wide range of severity within each DRG, but these differences did not always agree with the teaching classification or the Health Care Financing Administration's case-mix index. These findings suggest that patient classification by means of unadjusted DRGs does not adequately reflect severity of illness, and they indicate that prospective payment programs based on DRGs alone may unfairly and adversely discriminate against certain hospitals.

Centers for Medicare and Medicaid Services, U.S.↗