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Biomedical subjects

B E Miller

Publications and source records attributed to B E Miller.

At least 73 records · Page 4Linked to original sources
PubMed1990

Biochemical assay of Alzheimer's disease--associated protein(s) in human brain tissue. A clinical study.

The concentration of Alzheimer's disease-associated protein (ADAP) was measured in postmortem brain tissue samples of temporal or frontal cortex from 111 human brains using a sandwich immunoassay. Alzheimer's disease-associated protein has three major ALZ-50-reactive subunits, including A-68. This assay utilizes ALZ-50 and a rabbit antibody raised against a highly ADAP-enriched brain protein fraction. The frequently observed cross-reactivity of ALZ-50 with normal brain components in direct immunoassays is minimized by this configuration. There were 27 normal controls, 28 neurologic disease controls, and 56 Alzheimer's disease cases. The normal control and neurologic disease control cases had essentially no detectable level of ADAP, while ADAP was clearly detected in 85.7% of the Alzheimer's disease cases. Clinical dementia, neuritic plaques, and old age per se are not correlated with increased ADAP levels. This biochemical assay of ADAP may prove to be helpful as an adjunct in the clinicopathologic diagnosis of Alzheimer's disease.

Aged↗
PubMed1990

1,25-Dihydroxyvitamin D3 metabolism in a human osteosarcoma cell line and human bone cells.

The metabolism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a human osteoblastic sarcoma cell line, U-2 OS, and by primary cultures of human bone-derived cells was examined at physiologic (5 x 10(-11) M) and pharmacologic (3.5 x 10(-7) M) substrate concentrations. For metabolite identification purposes, cells nearing confluency were incubated for 18 h with 3.5 x 10(-7) M 1,25-(OH)2D3 in serum-free medium. The putative vitamin D metabolites produced during this incubation were isolated from a total lipid extract of cells and medium. Identification of the metabolites was achieved by comigration with authentic standards on three high-performance liquid chromatography systems, UV spectral analysis, mass spectrometry, and chemical modification by sodium borohydride and sodium metaperiodate. The identified metabolites produced from 1,25-(OH)2D3 by the human osteosarcoma cells include 1,24,25-trihydroxyvitamin D3; 24-oxo-1,25-dihydroxyvitamin D3; 24-oxo-1,23,25-trihydroxyvitamin D3; and 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3. Evidence is presented that (1) 1,25-(OH)2D3 metabolism occurs constitutively in U-2 OS osteosarcoma cells at a physiologic substrate concentration (5 x 11(-11) M), (2) the pathway can be further induced by pharmacologic 1,25-(OH)2D3 concentrations (10(-7) M), and (3) this pathway is present in primary cultures of normal human bone-derived cells.

Bone and Bones↗
PubMed1990

A sandwich enzyme immunoassay for detecting and measuring Alzheimer's disease--associated proteins in human brain tissue.

A simple, fast, and reliable immunoassay has been developed to detect and measure Alzheimer's disease-associated proteins (ADAPs) in human brain tissue. This assay, called ALZ-EIA (brain), was developed for Abbott's quantum system, in which 1/4-in. beads are used as solid phase. The beads were coated with polyclonal IgG purified from sera obtained from rabbits immunized with a highly enriched Alzheimer's disease brain protein fraction for ADAP. The antigen (ADAP) is effectively captured by the polyclonal IgG coated on the beads. ALZ-50 (a mouse monoclonal IgM) is used as the detection antibody, and bound ALZ-50 is subsequently quantified by a horseradish peroxidase-linked goat antimouse IgM and appropriate substrate. The assay is linear up to 0.5 absorbance units (r = 0.9), reproducible (CV less than 10%), sensitive, and specific. With preformulated reagents and standard supplies, the assay is simple and rapid; 120 data points can readily be generated in about 4 hr.

Alzheimer Disease↗
PubMed1990

Peak B endorphin concentration in cerebrospinal fluid: reduced in chronic pain patients and increased during the placebo response.

The level of an endogenous opioid (peak B endorphin) was measured in chromatographically fractionated cerebrospinal fluid (CSF) sampled from two groups of chronic pain patients before and after intrathecal saline (placebo) injection. As assessed by a verbal rating scale, one group reported no change in their level of pain (non-responders, NR; n = 6) while the other group reported complete or greater than 50% pain relief (placebo responders, PR; n = 14). We find, as has been reported previously, that initial peak B levels were lower (by 50%) in these chronic pain patients' CSF than in CSF from pain-free (PF) normal controls (P less than 0.001, t-test). Peak B levels measured from CSF of the NR group undergoing this procedure did not change (P greater than 0.4, paired t-test). In contrast, a significant 2.3-fold increase was measured in the CSF peak B level of the PR group (P less than 0.05, paired t-test). This is the first direct evidence that a CSF opioid is correlated with placebo pain relief in chronic pain patients. Peak B is a potent analgesic substance when administered by the intracerebroventricular route in mice and its level is related to the patients' pain status in a presumably causal manner.

Chronic Disease↗
PubMed1990

Detection of amyloid beta protein precursor immunoreactivity in normal and Alzheimer's disease cerebrospinal fluid.

The amyloid A4 (or beta protein), a 4.2 kD polypeptide, is a major component of amyloid deposits in the brains of patients with Alzheimer's Disease (AD). The self-aggregating amyloid A4 protein of AD is encoded as part of three larger proteins by the amyloid A4 precursor gene. The corresponding proteins have 695, 751 and 770 amino acid residues. To investigate the utility of amyloid beta protein precursor (A beta PP) as a diagnostic marker for AD an antiserum against a synthetic peptide (175-186), predicted from cDNA sequence for A beta PP, was used. The immunoreactivity of A beta PP in normal and AD cerebrospinal fluid (CSF) was measured by Western blot and detected with radiolabeled protein A. A total of fifty-seven CSF samples (AD = 27 and normal = 30) were analyzed for A beta PP immunoreactivity. A polyclonal antibody detected two major protein bands with apparent molecular weights of 105kD and 90kD both in normal and AD CSF. The difference between normal and AD CSF was not significant. These results indicate that immunoreactivity of A beta PP is present both in normal and AD CSF, and that the difference is too small to be used as a diagnostic marker.

Adult↗
PubMed1990

Induction of surfactant protein (SP-A) biosynthesis and SP-A mRNA in activated type II cells during acute silicosis in rats.

The synthesis of the major surfactant protein, SP-A, was studied in activated alveolar type II cells isolated from the lungs of rats exposed to silica by intratracheal instillation. Exposure of rats to silica resulted in large increases in the levels of disaturated phosphatidylcholine and SP-A in the extracellular and intracellular surfactant compartments. Isolated type II cells were used to determine if the observed increases in SP-A were associated with increased SP-A synthesis. Type II cells were isolated by a combination of elastase digestion, centrifugal elutriation, and differential adherence on IgG-coated petri dishes. Type II cells from silica-treated lungs were separated into two populations, designated type IIA and type IIB. The type IIB, or activated population, consisted of type II cells that were larger than normal type II cells and, in addition, contained larger and more numerous lamellar bodies than normal type II cells. Type IIB cells contained 4.3-fold higher levels of SP-A compared to normal type II cells. SP-A synthesis was measured by incubating freshly isolated cells with [35S]Translabel (70% [35S]methionine, 15% [35S]cysteine) for up to 4 h in methionine-free medium, followed by immunoprecipitation of newly synthesized protein. The rate of SP-A synthesis was increased approximately 6.7-fold in the activated type II cells. Analysis of the newly synthesized protein by one-dimensional SDS-PAGE indicated three intracellular forms of SP-A with molecular weights of approximately 26,000, 30,000, and 34,000. In type II cells from control rats, the 34-kD protein accounted for approximately 93% of the newly synthesized SP-A after 4 h of incubation; only a small amount of radioactivity was associated with the lower molecular weight species. The increased biosynthesis of SP-A in the activated type II cells was associated with a 7.3-fold increase in the level of SP-A mRNA. These results indicate that the content and synthesis of SP-A are both highly elevated in activated type II cells and that these increases may be due to increased levels of SP-A mRNA.

Animals↗
PubMed1990

Hypertrophy and hyperplasia of alveolar type II cells in response to silica and other pulmonary toxicants.

Alveolar Type II cells serve two major functions in the lung, both of which are essential for the preservation of normal lung function. First, Type II cells synthesize and secrete pulmonary surfactant, and second, they function as progenitor cells for maintaining the alveolar epithelium. The Type II cell population of the lung is quite sensitive to the deposition of toxicants in the distal lung, responding in two principal ways. Damage to the Type I epithelium stimulates Type II cells to proliferate and subsequently differentiate to replace the injured Type I cells. Second, a portion of the Type II cell population may become hypertrophic. Both of these events are frequent findings in the diseased or damaged lung. The Type II cell changes are often associated with increases in surfactant pools. In those cases where ultrastructural characteristics of hypertrophic Type II cells were examined, the appearance of these cells was consistent with that of an activated cell type. Alterations in the lamellar body compartment are a common finding in hypertrophic Type II cells, with increases in both lamellar body size and number. It is likely that the hypertrophic, or activated, Type II cells account for the increased levels of surfactant found in the lungs after exposure to a variety of toxic agents. We examined, in detail, Type II cell hyperplasia and hypertrophy induced by silica deposition. Both Type II cell hyperplasia and hypertrophy were prominent responses. The proliferative response led to an approximate doubling of the number of Type II cells in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Efficient recovery of clonogenic stem cells from solid tumors and occult metastatic deposits.

We describe the use of enzymes combined with brief, sequential mechanical disruptions in a Tekmar Stomacher blender for the recovery of clonogenic neoplastic cells from solid tumors, lungs, and livers. The method has yielded 3 X 10(8) to 5 X 10(8) total cells and 1.2 X 10(6) to 17 X 10(6) clonogenic cells per gram of tissue from three different mouse mammary tumor subpopulations growing in the subcutis. The clonogenic cell yields represent a 4- to 13-fold increase over our previous best method of tumor disaggregation. The increase in total cells recovered, while not as dramatic (up to 3-fold), was statistically significant for two of the three tumor lines. We were also able to efficiently recover 125I-iododeoxyuridine labelled neoplastic cells from lungs and livers after injecting the cells intravenously. Over half of the total radiolabel present in these organs prior to disaggregation could be recovered in the cell suspensions obtained.

Animals↗
PubMed1989

Growth regulation of mouse mammary tumor cells in collagen gel cultures by diffusible factors produced by normal mammary gland epithelium and stromal fibroblasts.

We have reported previously that both normal mammary epithelium and stroma stimulate the growth of mouse mammary tumors in vivo. We have devised a method to investigate the role of diffusible factors in these growth interactions in vitro. Because an appropriate matrix, a particular cell shape, or multicellular organization may be required for the production of factors and/or for the response to such factors, the method assesses the expansion of boluses of cells in collagen gel matrix. Under these culture conditions, paracrine effects were detected which were not observed when cells were growing in monolayer on plastic. The growth of mammary tumor lines 66, 410.4, and D2A1 was stimulated by both mammary epithelial cells and fibroblasts prepared from midpregnant mouse mammary glands. In contrast, these mammary tumor lines were inhibited by coculturing with normal mammary cells in monolayer on plastic. Stimulation in collagen and inhibition in monolayer cultures were both dose dependent; increasing numbers of regulator mammary cells increased the effects on tumor growth. Additional tumor cells did not stimulate growth of target tumor cell boluses in collagen gel cultures. This attempt to model the in situ situation may be a more appropriate model for the detection and characterization of relevant diffusible growth regulatory factors than monolayers on plastic and/or colonies growing in agar.

Animals↗
PubMed1989

Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase.

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.

Amino Acid Sequence↗
PubMed1989

Characterization of the cysJIH regions of Salmonella typhimurium and Escherichia coli B. DNA sequences of cysI and cysH and a model for the siroheme-Fe4S4 active center of sulfite reductase hemoprotein based on amino acid homology with spinach nitrite reductase.

The hemoprotein component of Salmonella typhimurium sulfite reductase (NADPH) (EC 1.8.1.2) was purified to homogeneity from cysJ266, a mutant strain lacking sulfite reductase flavoprotein. The siroheme- and Fe4S4-containing enzyme was isolated as a monomeric 63-kDa polypeptide and consisted of a mixture of unligated enzyme and a complex with sulfite. Following reduction with 5'-deazaflavin-EDTA and reoxidation, the complex was converted to the uncomplexed, high spin ferri-siroheme state seen previously with Escherichia coli sulfite reductase hemoprotein preparations. The S. typhimurium hemoprotein exhibited catalytic and physical properties identical to the hemoprotein prepared by urea dissociation of E. coli sulfite reductase holoenzyme and was fully competent in reconstituting NADPH-sulfite reductase activity when combined with excess purified sulfite reductase flavoprotein. The DNA sequences of cysI and cysH from S. typhimurium and E. coli B were determined and, together with previously reported data, confirmed the organization of this region as promoter-cysJ-cysI-cysH with all three genes oriented in the same direction from the promoter. Molecular weights deduced for the cysI-encoded sulfite reductase hemoprotein and for the cysH-encoded 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase were approximately 64,000 and 28,000, respectively. Comparison of the deduced amino acid sequence of sulfite reductase hemoprotein with that of spinach nitrite reductase (Back, E., Burkhart, W., Moyer, M., Privalle, L., and Rothstein, S. (1988) Mol. Gen. Genet. 212, 20-26), which also contains siroheme and an Fe4S4 cluster, showed two groups of cysteine-containing sequences with the structures Cys-(X)3-Cys and Cys-(X)5-Cys, which are homologous in the two enzymes and are postulated to provide the ligands of the Fe4S4 cluster in both proteins. From these sequences and from crystallographic (McRee, D. E., Richardson, D. C., Richardson, J. S., and Siegel, L. M. (1986) J. Biol. Chem. 261, 10277-10281) and spectroscopic data in the literature, a model is proposed for the structure of the active center of these two enzymes.

Amino Acid Sequence↗
PubMed1989

Therapeutic perturbation of the tumor ecosystem in reconstructed heterogeneous mouse mammary tumors.

We have measured the response to methotrexate in vivo of paired mixtures of sister subpopulation lines from a mouse mammary tumor, as a model of drug response of a heterogeneous tumor. The subpopulation lines differed in intrinsic sensitivity to methotrexate. Response was measured both as growth delay and as a shift in tumor cell population distribution toward the more resistant cell line. We found differences between two pairs of cell lines in growth delay: line 66 plus 4T07 mixtures tended to be as responsive as was line 4T07 (the more sensitive line) alone, whereas line 168 plus 4T07 mixtures tended to be less responsive than line 4T07 alone. With both paired mixtures, the tumors arising after treatment tended to contain more line 66 or line 168 than did untreated tumors, but this shift was extremely variable among individual tumors. Within most treatment groups, there was no correlation between the growth rate of individual mixed tumors and the final tumor cell distribution. Likewise, between experiments, there was no correlation between the amount of growth delay in mixed tumors and the final tumor cell distribution. Thus, the cellular composition of treated tumors did not directly reflect the response to therapy.

Animals↗
PubMed1989

Regulation of phosphatidylcholine biosynthesis in activated alveolar type II cells.

The biosynthesis of phosphatidylcholine was studied in a population of activated Type II cells isolated from the lungs of rats treated with silica. Type II cells were separated by centrifugal elutriation into two populations, designated Type IIA and Type IIB. The Type IIB or activated population consisted of Type II cells that were larger than normal cells; Type IIA cells were morphologically similar to normal Type II cells. Type IIB cells incorporated more [Me-14C]choline into both total phosphatidylcholine and disaturated phosphatidylcholine than did Type IIA or control Type II cells. Measurement of the pool sizes of the choline-containing precursors to phosphatidylcholine indicated that the biosynthesis of phosphatidylcholine was increased 4- to 5-fold in Type IIB cells. Increased conversion of cholinephosphate to CDP-choline was associated with increased phosphatidylcholine biosynthesis in Type IIB cells. Cholinephosphate cytidylyltransferase activity was increased approximately threefold in Type IIB cells. Subcellular fractionation indicated that essentially all of the increase in cytidylyltransferase activity was associated with the particulate fraction (100,000 x g pellet). In Type IIB cells, the particulate fraction contained 83% of the total cellular cytidylyltransferase activity; in control cells, this fraction contained 67% of the total activity. The specific activity of the cytidylyltransferase associated with the particulate fraction was increased twofold in Type IIB cells. The specific activity of the cytosolic enzyme was similar to that in control cells. Cholinephosphotransferase specific activity was increased approximately twofold in the activated Type II cells. The specific activity of choline kinase was the same as that in control Type II cells. These results demonstrate that the increased biosynthesis of phosphatidylcholine in Type IIB cells is a result of stimulation of the CDP-choline pathway. This study indicates that both cholinephosphate cytidylyltransferase and cholinephosphotransferase may be involved in regulating the de novo biosynthesis of phosphatidylcholine in alveolar Type II cells.

Animals↗
PubMed1989

Therapeutic implications of tumor heterogeneity.

At the outset of this review, we stated that we wished to raise some questions that challenge the commonly held view that tumor heterogeneity is of major significance to treatment failure. The nature of this challenge is the following: although tumor heterogeneity in sensitivity to therapeutic agents has been demonstrated repeatedly, using isolated subpopulations of cells, primarily in cell culture systems, there is very little work that has been directed toward asking the tough questions about how that heterogeneity actually impacts on the response to treatment in vivo. In our own work, when we have attempted to simulate heterogeneity, in vivo or in vitro with mixed populations of tumor cells, we have seen that the simple prediction that treatment response would reflect the sensitivities of the individual subpopulations was not valid. Tumor subpopulation interactions, influencing both growth and drug sensitivity, resulted in treatment responses that were either better or worse than would be expected. Shifts in the distribution of subpopulations under the influence of therapy did not necessarily correlate with treatment response. Marked differences in the relative proportions of subpopulations within tumors did not necessarily translate into marked differences in the behavior of whole tumors. Imposition of in vivo-like three-dimensional tissue architecture caused major changes in the overall drug sensitivity of individual subpopulations, beyond those seen as a result of heterogeneity. Of course we realize that our work is very limited, one tumor system and a few treatment protocols, but that is the challenge. Much more in-depth experimental and clinical research is necessary in order to evaluate how, and how much, tumor heterogeneity really does affect treatment. Without such work, efforts to devise more effective treatment strategies, based on common assumptions and theoretical models, rather than experimental analysis, can only be superficial and, ultimately, useless.

Animals↗
PubMed1988

Dominance of a tumor subpopulation line in mixed heterogeneous mouse mammary tumors.

When mixtures of cell lines 168 and 4T07, both derived from the same mouse mammary tumor, were injected into syngeneic mice, the resulting tumors, analyzed over a large size range by colony-forming assays in selective media, consisted primarily of line 4T07, even when the ratio injected was 100:1 or greater in favor of line 168. This result indicated a suppression of growth of line 168, since the volume-doubling time of line 168 tumors in the absence of line 4T07 was one-half that of line 4T07 tumors. That growth suppression was not due to inhibition of line 168 by immunity induced to line 4T07 was shown in two ways: (a) line 168 tumors grew almost as well in mice preimmunized with line 4T07 as in controls, whereas line 4T07 tumor growth was strongly inhibited in preimmunized mice; and (b) the final composition (favoring line 4T07) in mixed tumors was similar in tumors grown in mice immunosuppressed by irradiation to that in nonirradiated controls. The strong suppression of line 168 did not occur when the two cell lines were injected simultaneously at different s.c. sites, nor did it occur when line 168 cells were injected in mixtures with lethally irradiated line 4T07 cells. Line 4T07 cells also suppressed the growth of line 168 cells in monolayer cultures. It was not likely that suppression was due to competition for growth factors, since the effect required cell contact. Suppression probably was not mediated through junctional communication, since these cells do not engage in metabolic cooperation. We suggest that a growth-inhibitory factor produced by line 4T07 mediates the suppression of 168 cells.

Animals↗
PubMed1988

Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica.

The effects of intratracheally instilled silica (10 mg/rat) on the biosynthesis of surfactant phospholipids was investigated in the lungs of rats. The sizes of the intracellular and extracellular pools of surfactant phospholipids were measured 7, 14 and 28 days after silica exposure. The ability of lung slices to incorporate [14C]choline and [3H]palmitate into surfactant phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) was also investigated. Both intra- and extra-cellular pools of surfactant phospholipids were increased by silica treatment. The intracellular pool increased linearly over the 28-day time period, ultimately reaching a size 62-fold greater than controls. The extracellular pool also increased, but showed a pattern different from that of the intracellular pool. The extracellular pool increased non-linearly up to 14 days, and then declined. At its maximum, the extracellular pool was increased 16-fold over the control. The ability of lung slices to incorporate phospholipid precursors into surfactant-associated PC and DSPC was elevated at all time periods. The rate of incorporation of [14C]choline into surfactant PC and DSPC was maximal at 14 days and was nearly 3-fold greater than the rate in controls. The rate of incorporation of [3H]palmitate was also maximal at 14 days, approx. 5-fold above controls for PC and 3-fold for DSPC. At this same time point, the microsomal activity of cholinephosphate cytidylyltransferase was increased 4.5-fold above controls, but cytosolic activity was not significantly affected by silica treatment. These data indicate that biosynthesis of surfactant PC is elevated after treatment of lungs with silica and that this increased biosynthesis probably underlies the expansion of the intra- and extra-cellular pools of surfactant phospholipids.

Animals↗
PubMed1988

Spontaneous fusion between metastatic mammary tumor subpopulations.

This study describes a differential frequency of spontaneous fusion between metastatic and nonmetastatic subpopulations derived from a single mouse mammary tumor. Subpopulations 66, 66c14 (a variant of 66 which is resistant to both thioguanine and ouabain), 410.4, and 44FTO (a thioguanine-resistant, ouabain-resistant derivative of 410.4) spontaneously metastasize from subcutaneous and mammary fatpad sites. Subpopulations 168, 168FARO (a diaminopurine-resistant, ouabain-resistant derivative of 168), 67, 68H, and 410 do not. The ability of these subpopulation lines to fuse spontaneously in vitro was determined after coculturing a drug-resistant line with a wild-type line in nonselective media. After 16-20 h of coculture, cells were plated in the appropriate media to select for fusion products--either HAT (hypoxanthine, aminopterin, thymidine) plus ouabain or AA (alanosine, adenine) plus ouabain--to determine the number of colony-forming cells (fusion products) present per 10(4) cells plated. When both subpopulations of the pair in the fusion mixture were metastatic, a significantly greater number of fusion products was recovered than if one or both of the subpopulations in the fusion mixture was nonmetastatic, with one exception: line 410 readily fused with both 66c14 and 44FTO. Subline 410 was highly metastatic when originally isolated but lost its metastatic competence after a brief time in tissue culture.

Animals↗