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Biomedical subjects

A F Chambers

Publications and source records attributed to A F Chambers.

At least 127 records · Page 7Linked to original sources
PubMed1985

Quantitative genetic analysis of tumor progression.

Metastasis and resistance to chemotherapy are common features of progressed cancers. With respect to the latter phenotype, it is thought that during tumor growth drug-resistant cells arise spontaneously at rates characteristic of the genetic alterations involved. On application of chemotherapy, such variant tumor cells are more likely to survive, and they may eventually dominate, resulting in a non-responsive malignancy. Aspects of this model have been confirmed in a number of experimental systems and in patients. In contrast to our understanding of drug resistance, steps involved in the progression to metastatic spread of tumor cells are much less well-understood. In this review we describe methodologies of quantitative genetic analysis with reference to development of drug resistance. We then describe attempts by ourselves and others to use a similar approach to investigate metastatic properties. Based on these studies, we have proposed the quantitative 'dynamic heterogeneity' model of tumor metastasis, which is presented here. Using an 'experimental' metastasis assay and Luria-Delbruck fluctuation analysis, we determined that in murine KHT fibrosarcoma and B16 melanoma lines, 'metastatic' variants with a distinct phenotype are generated at high rates. These variants are relatively unstable resulting in a dynamic equilibrium between generation and loss of metastatic variants. The metastatic ability of such a tumor population is thus dependent on the frequency of a subpopulation of metastatic variants which are turning over rapidly. This dynamic heterogeneity model is able to quantitatively provide a unifying explanation for a wide range of observations concerning tumor heterogeneity and clonal instability. Genetic mechanisms involving rapid rates have been characterized in drug-resistant variants. We speculate that similar processes may be involved in different aspects of tumor progression such as those resulting in metastasis.

Animals↗
PubMed1985

Cells transformed with a ts viral src mutant are temperature sensitive for in vivo growth.

Studies on ts mutants of avian sarcoma viruses have previously implicated the src gene product (pp60src) kinase function in in vitro transformation. The role of src in vivo, however, has not been clearly defined. Using a sensitive and quantitative assay that was developed in chicken embryos (Chambers et al., Cancer Res. 42:4018-4025, 1982), we tested the in vivo tumorigenic properties of cells transformed with LA23, an avian sarcoma virus that is temperature sensitive for in vitro transformation. We found that the in vivo growth ability of these cells was temperature sensitive and that this in vivo behavior correlated with the in vitro transformation behavior (growth in soft agar and saturation density).

Animals↗
PubMed1985

Severity of illness within DRGs: impact on prospective payment.

This study compares the financial impact of a Diagnosis Related Group (DRG) prospective payment system with that of a Severity of Illness-adjusted DRG prospective payment system. The data base of about 106,000 discharges is from 15 hospitals, all of which had a Health Care Financing Administration (HCFA) DRG case mix index greater than 1. In order to pool the data over the 15 hospitals, all charges were converted to costs, normalized to Fiscal Year 1983, and adjusted for medical education and wage levels. The findings showed that, for the study population as a whole, DRGs explained 28 per cent of the variability in resource use per case while Severity of Illness-adjusted DRGs explained 61 per cent of the variability in resource use per case. When we simulated prospective payment systems based on DRGs and on Severity-adjusted DRGs, we found that the financial impact of the two systems differed by very little in some hospitals and by as much as 35 per cent of total operating costs in other hospitals. Thus, even with a data set that is relatively homogeneous (with respect to the HCFA DRG case mix index definition of hospitals), we found substantial inequities in payment when DRGs were not adjusted for Severity of Illness. These findings suggest that, with a more representative set of hospitals, the difference between unadjusted and Severity-adjusted DRG-based prospective payment could be greater than 35 per cent of a hospital's total operating costs.

Costs and Cost Analysis↗
PubMed1985

Experimental metastatic ability of H-ras-transformed NIH3T3 cells.

We have used a quantitative "experimental" metastasis assay in the embryonic chick, an immunodeficient host, to examine in vivo growth properties of ras oncogene-transformed NIH3T3 cells. We found that two independently derived populations of NIH3T3 cells that had been morphologically transformed with the T24 human H-ras oncogene were able to grow in vivo following i.v. injection. Nontransformed control NIH3T3 cells with normal morphology did not grow in this assay. Spontaneously arising morphological transformants from control NIH3T3 cell populations were also tested and did not grow in this assay. We conclude that the H-ras gene can confer experimental metastatic ability on nonmetastatic NIH3T3 cells, that the ras gene alters the cells in some way beyond in vitro morphological transformation, and thus that the in vitro transformation assay detects only part of the malignant phenotype of these cells.

Animals↗
PubMed1984

Dynamic heterogeneity: rapid generation of metastatic variants in mouse B16 melanoma cells.

The ability of clonally derived lines of B16F1 and B16F10 melanoma cells to form experimental metastases in C57BL mice after intravenous injection was examined. Luria- Delbruck fluctuation analysis was applied to the results obtained with parallel subclones grown to small population sizes before testing for metastatic ability. The analysis demonstrated that variant cells capable of forming experimental metastases were generated in B16F1 cell populations at an effective rate of about 1.3 X 10(-5) per cell per generation while in B16F10 cell populations the effective rate of production was about 5 X 10(-5) per cell per generation. These results are consistent with a dynamic heterogeneity model of tumor progression. They suggest that the majority of cells in both lines are effectively nonmetastatic and that the higher metastatic ability of the B16F10 population may be due in part to a higher rate of generation of metastatic variants.

Animals↗
PubMed1984

Dynamic heterogeneity and metastasis.

Parallel clonal populations grown to small defined sizes were used to quantitate rates of generation of metastatic cells. In murine KHT fibrosarcoma and B16 melanoma lines, metastatic cells are generated at effective rates of 10(-5) per cell per generation, or greater. These variant cells are unstable, and are apparently lost at very high rates. It thus appears that metastases could arise from unstable variants, and that rapid rates of change in some phenotypes may be an important feature of malignant progression. We have called such rapid variations dynamic heterogeneity. This may be a useful concept for further investigating aspects of tumor heterogeneity.

Animals↗
PubMed1984

Rapid phenotype variation in cells derived from lung metastases of KHT fibrosarcoma.

We have established previously that intravenously derived metastatic variants are generated in KHT fibrosarcoma cells at an effective rate of 10(-5)/cell/generation. To study the properties of these variants further, we examined several lines of KHT fibrosarcoma cells obtained from experimental lung metastases. When tested using an experimental metastasis assay, some of the lines were highly metastatic, relative to parental lines, but these highly metastatic phenotypes were often rapidly lost as the lines were grown in vitro, and both decreases and increases in metastatic ability were observed. In another set of experiments, lines obtained by 10 serial selections of experimental lung metastases without intervening in vitro growth between passages were also analyzed. Again, while highly metastatic phenotypes were observed in some instances, they did not persist beyond 1 or 2 in vivo passages, and the series as a whole failed to reveal a persistent increase in ability to form experimental metastases. We conclude from these experiments that although metastatic variants are generated at high rates in KHT cell lines, the phenotype is lost at even higher rates, and metastatic variants represent only a small proportion of the tumor cell population. Thus, it appears that in this system rapid phenotypic variation may play an important role in the metastatic process.

Animals↗
PubMed1984

Selection for experimental metastatic ability of heterologous tumor cells in the chick embryo after DNA-mediated transfer.

The chick embryo is an immune-deficient host able to support growth of a wide variety of transformed cells. Since growth of normal cells is not observed, this system appears to be generally useful for investigating malignant properties of different cells. Recently, we developed a sensitive assay to quantitate and select for rodent cells able to survive and grow in embryonic chick organs following i.v. injection (Cancer Res., 42: 4018-4025, 1982). We envisage this assay as a model system for studying aspects of the metastatic process. We have used DNAs from murine and human melanoma cell lines (which grow well in chick embryos after i.v. injection) to transfect murine LTA cells (which do not grow in chicks after i.v. injection). From the transfected LTA cells, we were able to isolate clones which grow well in the chick after i.v. injection. Such clones were not observed in untransfected LTA cells or with LTA cells transfected with LTA DNA. These experiments clearly demonstrate the feasibility of using the chick embryo as a host system to study genes involved in growth control alteration of the sort seen in malignant transformation.

Animals↗
PubMed1982

Metastatic variants are generated spontaneously at a high rate in mouse KHT tumor.

Using the Luria-Delbrück fluctuation analysis, we have examined the lung tumor-forming ability of a series of parallel clones derived from the KHT tumor, grown to small defined sizes. From these studies, we conclude that metastatic variants arise spontaneously in the clonal lines during their growth, at an apparent rate of approximately 10(-5) per cell per generation. This rapid rate has implications for our understanding of tumor heterogeneity and the process of tumor progression. Previous results have suggested that heterogeneity observed in cloning experiments reflects stable subpopulations of cells in the original tumor. We propose here an alternative "dynamic heterogeneity" model, in which metastatic variants arise at a high rate (as detected in the cloning experiments) but need not be stable mutations in order to effectively produce metastases.

Animals↗
PubMed1982

A model system for studying metastasis using the embryonic chick.

An assay capable of recovering individual viable rodent cells localized in various organs of the chick embryo is described. This assay is based on the differential sensitivity of chick and rodent cells to the cytotoxic drug ouabain. Utilizing this assay, the potential of the chick embryo as a model system for studying metastasis was examined. Several cell lines were characterized in three ways: (a) ability to form local tumors after cell application onto the chorioallantoic membrane; (b) ability to form macro- or microscopic metastasis in the embryo from chorioallantoic membrane tumors; (c) experimental metastatic ability following i.v. injection into chorioallantoic membrane veins. These results were compared with the results obtained from the ouabain-plating assay. We conclude that this assay permits detection of viable metastatic cells even when tumors cannot be detected and helps to overcome the time constraints that have, in the past, limited the usefulness of the chick embryo in modeling metastasis.

Allantoin↗
PubMed1981

Tumor heterogeneity and stability of the metastatic phenotype of mouse KHT sarcoma cells.

Heterogeneity in metastatic ability has been demonstrated in model systems for in vitro-cloned cell lines for a number of different tumors. We have examined the clonal diversity of mouse KHT sarcoma cells cloned either in vitro or in vivo by determining their ability to form lung colonies following i.v. injection into syngeneic mice. A wide range of metastatic ability was found in both the in vitro- and in vivo-isolated clones, suggesting that the diversity observed is not due to any selection occurring during in vitro growth. The stability of four in vitro-isolated clones, two of high metastatic and two of low metastatic ability, was then studied over a period of 3 to 4 months of growth in vitro. The phenotype of the highly metastatic cells remained relatively stable, declining only slightly over time. The clones with low metastatic ability, however, demonstrated a significant increase in ability to form lung colonies over the first 30 days in culture before becoming stable at levels approximately 10-fold higher than their original values. Even after this increase, however, there remained a difference of about a factor of 10 in the metastatic ability of the high and low pairs of clones. It was found that the number of lung colonies formed by all four cell lines was significantly increased when plastic microspheres were injected with the cell suspension. The cells with low metastatic ability were affected to a greater degree by the microspheres, resulting in the elimination of the difference between the four clones after 30 days in culture. This result suggests that the use of microspheres may provide a means to distinguish different cellular properties which affect the ability of cells to form lung metastases.

Animals↗
PubMedDate not supplied

ras transfection and expression does not induce progression from tumorigenicity to metastatic ability in mouse LTA cells.

Studies testing the ability of a transfected ras oncogene to confer metastatic properties on non-metastatic cells have yielded conflicting results. Most of these studies have used recipient cells at early stages of progression (primary or immortalized, non-tumorigenic lines). In this study we tested the ability of the T24-H-ras oncogene to induce progression of tumorigenic, non-metastatic, murine LTA cells to a metastatic phenotype. Metastatic ability was assessed in complementary assays in two immune-deficient hosts, nude mice (after s.c. injection) and chick embryos (after i.v. injection), to determine if ras transfection affected metastatic properties in hosts lacking an intact immune system. Even with greatly elevated levels of ras p21 protein, pools of ras-transfected cells as well as individual clonal populations remained non-metastatic in both hosts. Serial in vivo passaging did not consistently enhance for either ras expression or metastatic ability. We conclude that expression of an activated ras oncogene in LTA cells does not induce progression from a tumorigenic to a metastatic phenotype. These results are in marked contrast to those obtained for ras expression in most other types. High levels of expression of an activated ras oncogene thus do not always promote progression from tumorigenicity to metastatic ability.

Animals↗
PubMedDate not supplied

Modulation of clonal progression in B16F1 melanoma cells.

We have examined the effects of the microenvironment on the frequency and generation of metastatic variant cells in both parental B16F1 melanoma cells and nascent clones. The metastatic abilities of cultured B16F1 cells were tested after a period of growth in the presence or absence of a second cell population separated from each other by a transwell membrane (0.45 micron pore size). The first population is defined as the 'responder' cells and the second as the 'stimulator' cells. We found that the presence of 10(5) B16F1 stimulator cells during the growth of responder B16F1 cells from approximately 10(4) to approximately 10(6) cells resulted in cells with an increased metastatic phenotype (greater than 8-fold increase in median number of lung tumors relative to untreated B16F1 parental cells). The presence of stimulator cells also increased the metastatic phenotype of nascent clones, which were grown to a population size of less than 10(6) cells, suggesting that the rate of generation of metastatic variants of the responder B16F1 clones was affected by the stimulator cells. Other cell lines, including highly metastatic B16F10 and BL6 melanoma cells, and KHT35-L1 fibrosarcoma cells, were effective stimulator cells when as few as 10(4) cells were added to transwells. In addition, normal immortalized NIH 3T3 cells were effective stimulator cells only at 10(5) cells/transwell. The cell density at which untreated parental B16F1 cells were harvested (3 x 10(3)-3 x 10(5) cells/cm2) did not affect the median number of lung tumors significantly. These results suggest that factors released from both tumor and immortalized normal cells can modulate epigenetic changes in the metastatic phenotype of B16F1 melanoma cells.

Animals↗
PubMedDate not supplied

Use of NeoR B16F1 murine melanoma cells to assess clonality of experimental metastases in the immune-deficient chick embryo.

Recent work on molecular and genetic aspects of metastasis has emphasized the need for assays in immune-deficient animal hosts. The commonly used assays in athymic nude mice may not always be appropriate, and assays in other hosts are required. We have developed a metastasis assay in the naturally immune-deficient chicken embryo. As part of our characterization of this assay we have examined the clonality of individual experimental (i.v.-derived) metastases in this host. For these studies we developed a cell line, B16-Neo, from parental B16F1 murine melanoma cells. B16-Neo cells carry a stable drug-resistance marker, the bacterial neo gene, which confers resistance to the drug G418, but are unaltered in experimental metastatic properties in the chick embryo relative to parental B16F1 cells. We observe that the majority of individual liver tumors that arise following i.v. injection of mixtures of these cells contain cells of a single marker phenotype and are likely to be clonal in origin. These results are similar to those obtained by others for metastases in immune-competent mice, suggesting similar mechanisms of metastasis formation in these two systems. In both hosts it should be noted, however, that a small but significant proportion of metastases appear not to be clonal in origin.

Animals↗
PubMedDate not supplied

Preclinical assessment of anti-cancer therapeutic strategies using in vivo videomicroscopy.

Preclinical in vivo studies of agents targeted against metastasis have to date been based primarily on end-point assays. Such assays can determine whether a treatment affects the number or size of metastases in an organ at a given time, but are poorly suited to determining how and at what stage in the process the treatment affected the end point. High resolution in vivo videomicroscopy permits direct observation of the process of metastasis as it occurs in living animals over time. Studies based on this technique and a cell accounting procedure we have devised, have shown that early steps in the metastatic process (survival in the circulation, extravasation) contribute relatively little to cell loss and metastatic inefficiency. Steps that occur after extravasation appear to be primarily responsible for the significant losses that result in metastatic inefficiency, and these steps may represent good targets for the design of new antimetastatic therapies. Matrix metalloproteinases have been implicated functionally in metastasis, and are viewed as an appropriate target in the development of inhibitors of metastasis. Using both endogenous and synthetic exogenous metalloproteinase inhibitors, we have shown that the inhibition of metastasis which these agents produce is not due to inhibition of cell extravasation from the circulation into the tissue, but to reduction of angiogenesis within metastases. A similar conclusion was reached concerning the mechanism of action, on metastasis, of carboxyamidotriazole, an inhibitor of calcium-mediated signal transduction which is currently in Phase II single agent clinical trials. In vivo videomicroscopy of sequential steps in metastasis, coupled with methods that allow precise quantification of cell loss at specific steps in the metastatic process, as well as standard histological assessment at stages identified as crucial, allow characterization of the details of metastasis as an ongoing process. This provides a powerful complement to end-point assays, for it allows mechanistic information to be obtained from in vivo experiments, an approach which provides better understanding of how and when a drug may function in vivo to inhibit metastasis.

Animals↗
PubMedDate not supplied

Induction of expression of osteopontin (OPN; secreted phosphoprotein) in metastatic, ras-transformed NIH 3T3 cells.

We have previously shown that transfection of NIH 3T3 cells with the T24 H-ras oncogene converts the cells to a tumorigenic and metastatic phenotype, in proportion to levels of ras expression. We hypothesize that ras-induced increases in malignancy occur via altered expression of various genes. We have identified OPN (osteopontin; also known as Secreted Phosphoprotein, 2ar, Eta-1, and transformation-associated phosphoprotein) as a ras-induced gene in these cells. We report here that expression of OPN RNA and secretion of OPN protein are increased in a series of ras-transformed NIH 3T3 cells, in proportion to levels of expression of ras. Detection of secreted OPN protein was facilitated by a barium citrate precipitation procedure. Although the function of this protein in tumor cells is not known, OPN contains a conserved GRGDS (glycine-arginine-glycine-aspartic acid-serine) amino acid sequence, which may function as a cell attachment site for this protein. We speculate that increased expression of OPN contributes to the increased malignancy of ras oncogene-transformed NIH 3T3 cells, perhaps by alterations in either adhesive properties or integrin-mediated signal transduction pathways.

3T3 Cells↗