Cell Line Model System Research Articles

ATR functions as a gene dosage-dependent tumor suppressor on a mismatch repair-deficient background.

The ataxia-telangiectasia mutated and rad3-related (ATR) kinase orchestrates cellular responses to DNA damage and replication stress. Complete loss of ATR function leads to chromosomal instability and cell death. However, heterozygous ATR mutations are found in human cancers with microsatellite instability, suggesting that ATR haploinsufficiency contributes to tumorigenesis. To test this possibility, we generated human cell line and mouse model systems in which a single ATR allele was inactivated on a mismatch repair (MMR)-deficient background. Monoallelic ATR gene targeting in MLH1-deficient HCT 116 colon carcinoma cells resulted in hypersensitivity to genotoxic stress accompanied by dramatic increases in fragile site instability, and chromosomal amplifications and rearrangements. The ATR(+/-) HCT 116 cells also displayed compromised activation of Chk1, an important downstream target for ATR. In complementary studies, we demonstrated that mice bearing the same Atr(+/-)/Mlh1(-/-) genotype were highly prone to both embryonic lethality and early tumor development. These results demonstrate that MMR proteins and ATR functionally interact during the cellular response to genotoxic stress, and that ATR serves as a haploinsufficient tumor suppressor in MMR-deficient cells.

Identification of Wnt responsive genes using a murine mammary epithelial cell line model system.

BackgroundThe Wnt/Wg pathway plays an important role in the developmental program of many cells and tissues in a variety of organisms. In addition, many Wnts and components of their downstream signaling pathways, such as β-catenin and APC, have been implicated in tumorigenesis. Over the past years, several genes have been identified as Wnt responsive, including c-myc, siamois, and cyclin D1.ResultsIn order to identify additional genes responsive to Wnt signaling that contribute to the transformed phenotype, we performed a cDNA subtractive hybridization screen between a mouse mammary epithelial cell line that overexpresses Wnt-1 (C57MG/Wnt-1) and the parental cell line (C57MG). The screen identified a total of 67 genes to be up-regulated in response to Wnt signaling. Of these 67 genes, the up-regulation of 62 was subsequently confirmed by Northern and dot blot analyses (and, for a subset, semi-quantitative PCR) of RNA isolated from C57MG cells subjected to (1) an independent Wnt-1 retroviral infection, and (2) co-culture with Wnt-1 expressing cells. Among the confirmed Wnt-1 responsive genes, we further characterized a mouse homolog of the human transcription factor Basic Transcription Element Binding protein 2 (BTEB2), Wnt-1 Responsive Cdc42 homolog (Wrch-1), and Wnt-1 Induced Secreted Protein (WISP-1).ConclusionSeveral novel genes were identified in this screen, as well as others that have been shown previously to be regulated by Wnt signaling, such as connexin43. The results indicate that cDNA subtractive hybridization is a useful method for identifying genes involved in the process of Wnt-1-induced transformation.

Amplification of a 280-Kilobase Core Region at the ERBB2 Locus Leads to Activation of Two Hypothetical Proteins in Breast Cancer

Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.

A Novel Fluorometric Invasion Assay for Quantitative Determination of Tumor Cell Invasion

T umor cell invasion into the basement membrane is an important step in the multistep process of cancer metastasis (Liotta, 1986). Various in vitro assay systems have been developed for the investigation of tumor cell invasion into the basement membrane. These methods primarily employ a reconstituted basement membrane matrix (Matrigel) as an invasive substrate coated on a microporous filter membrane within a Boyden chamber (Albini et al, 1987; Schlechte et al, 1990). In such systems, establishing a uniform horizontal layer of Matrigel is difficult. In addition, after careful removal and fixation of the filter, counting the number of invaded cells under a high-powered microscope is time consuming, laborious, and sometimes subjective. To overcome such disadvantages, we devised an invasion 3-(4, 5-Dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay with a uniform Matrigel layer prepared by a water-repellent treatment with paraffin and a tetrazolium-based colorimetric MTT assay for counting the invaded cells (Imamura et al, 1994). However, with this procedure, accurate quantitation of cell invasion requires the careful removal of noninvaded cells from the surface of the Matrigel layer using a cotton swab. We then developed a fluorometric invasion assay for quantitative determination of tumor cell invasion that uses a fluorescence-blocking membrane insert (Falcon HTS FluoroBlok insert, Becton Dickinson Labware, Franklin Lakes, New Jersey), as well as a uniform Matrigel layer produced by a paraffin-treated water-repellent method. The fluorescence-blocking microporous membrane was developed by Tchao (1997). In this assay cells are fluorescently labeled with a 3', 6'-(Di (O-acetyl)-2', 7'-bis[N, N-bis (carboxymethyl) aminomethyl]) fluorescein, tetraacetoxymethyl ester (calcein-AM) (Dojindo Lab., Kumamoto, Japan) in the invasion assay chamber at the end of the experimental period, thus avoiding potential cytotoxicity problems over the long incubation period of the assay. The number of invaded cells can be quantified, with no requirement for removing the noninvaded cells, through the use of a fluorescence-blocking membrane, which blocks almost all of the fluorescence apart from a very low level of background through the micropores. For water-repellent treatment, a block of paraffin was rubbed on the inside wall of the insert and residue was removed with a cotton swab. The water-repellent–treated FluoroBlok inserts were sterilized with ethylene oxide gas and placed into 24-well tissue culture plates (FIA chamber). For Matrigel coating, 70 l of 0.2 mg/ml Matrigel was applied to the membrane filters of the insert. Filters were dried overnight (for more than 12 hours) in a laminar flow hood. The outline of our assay is shown in Figure 1. Conditioned medium (700 l), obtained by incubating NIH3T3 cells for 24 hours in serum-free medium (Ajinomoto Co., Tokyo, Japan) was then placed in the lower well as a chemoattractant. Culture media (200 l) was gently added to the upper well to reconstitute the Matrigel layer and to provide growth medium for the cells. Finally the FIA chamber with medium (inner) and attractant (outer) was incubated for 60 minutes before cell addition. To the inner well, 100 l of cell suspension resuspended in culture medium at a density of 1 10 cells/ml (1 10 cells) was added. After incubation for 72 hours, the FIA chamber was gently rinsed with PBS to wash out the remaining serum. Subsequently 700l and 300l aliquots of calcein-AM solution at a concentration of 2 g/ml in PBS were added into the outer and inner wells, respectively. After a 60-minute incubation, the calcein-AM solution was exchanged with PBS to reduce the background fluorescence. The fluorescence in the FIA chamber was measured immediately from the bottom of the plates using a Cytofluor4000TC fluorescent multiwell plate reader (PerSeptive Biosystems, Framingham, Massachusetts) using an excitation wavelength of 485 nm and an emission wavelength of 530 nm (gain is 50). Received October 18, 2001. This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, and the Ministry of Health and Welfare, Japan. Address reprint requests to: Dr. Hiroyoshi Yoh, The First Department of Surgery, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: yohdr@po.synapse.ne.jp 0023-6837/02/8201-109$03.00/0 LABORATORY INVESTIGATION Vol. 82, No. 1, p. 109, 2002 Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A.

Nonpeptidomimetic farnesyltransferase inhibitor RPR-115135 increases cytotoxicity of 5-fluorouracil: role of p53.

A new nonpeptidic farnesyltransferase inhibitor, RPR-115135, in combination with 5-fluorouracil (5-FU) was studied in an isogenic cell line model system consisting of human colon cancer HCT-116 cells. HCT-116 cells were transfected with an empty control pCMV vector and with a dominant-negative mutated p53 transgene (248R/W). We found that, relative to control transfectants, there was a slight tendency for the p53 inactivated cells to be less sensitive to 5-FU after 6 days of continuous treatment. Simultaneous administration of RPR-115135 and 5-FU, at equitoxic concentrations, resulted in an enhancement of 5-FU cytotoxicity, especially in the CMV-2 clone. Growth inhibition could be accounted for on the basis of a specific cell cycle arrest phenotype (G(2)-M arrest in CMV-2 and S arrest in mutated clones), as assayed by flow cytometry. The combination RPR-115135 + 5-FU increases apoptotic events only in the CMV-2 clone.

Down-regulation of N-type voltage-activated Ca2+ channels by gabapentin.

1. Although the cellular and molecular mechanisms of the anticonvulsant action of gabapentin (GBP) remain incompletely described, in vitro studies have shown that GBP binds to the alpha2delta subunit of the high voltage-activated (HVA) Ca2+ channels. 2. In this report, we analyzed the effects of GBP on the functional expression of HVA Ca2+ channels in the PC12 cell line model system. Negligible inhibition of Ca2+ channel activity was observed after acute treatment, but a significant decrease in Ca2+ current amplitude was promoted by chronic exposure to GBP. 3. Consistent with this, radioligand binding experiments showed a comparable reduction in the total number of membrane HVA N-type channels after GBP treatment.

From chromosomal alterations to target genes for therapy: integrating cytogenetic and functional genomic views of the breast cancer genome

A vast number of recurrent chromosomal alterations have been implicated in cancer development and progression. However, most of the genes involved in recurrent chromosomal alterations in solid tumors remain unknown, despite the recent substantial progress in genomic research and availability of high-throughput technologies. For example, it is now possible to quickly identify large numbers of differentially expressed genes in cancer specimens using cDNA microarrays. Integration of this ‘functional genomic view’ of the cancer genome with the ‘cytogenetic view’ could lead to the identification of genes playing a critical role in cancer development and progression. In this review, we illustrate how the combination of three different microarray technologies, cDNA, CGH, and tissue microarrays, makes it possible to directly identify genes involved in chromosomal rearrangements in cell line model systems and then rapidly explore their significance as potential diagnostic and therapeutic targets in human primary breast cancer progression.

Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells.

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.

Cellular effects of a new farnesyltransferase inhibitor, RPR-115135, in a human isogenic colon cancer cell line model system HCT-116.

Annals of the New York Academy of SciencesVolume 886, Issue 1 p. 252-256 Cellular Effects of a New Farnesyltransferase Inhibitor, RPR-115135, in a Human Isogenic Colon Cancer Cell Line Model System HCT-116 P. RUSSO, Corresponding Author P. RUSSO Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA Laboratory of Experimental Oncology, National Cancer Institute and Department of Clinical and Experimental Oncology, University of Genoa, Genoa I-16132, Italy To whom correspondence should be addressed: Department of Experimental Oncology, National Cancer Institute, Largo Rosanna Benzi 10, I-16132 Genoa, Italy. Phone, +39 010 5600212; fax, +39 010 5600210. e-mail, [email protected]Search for more papers by this authorC. OTTOBONI, C. OTTOBONI Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA Laboratory of Experimental Oncology, National Cancer Institute and Department of Clinical and Experimental Oncology, University of Genoa, Genoa I-16132, ItalySearch for more papers by this authorC. FALUGI, C. FALUGI Department of Developing Biology, University of Genoa, Genoa, ItalySearch for more papers by this authorW. REINHOLD, W. REINHOLD Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USASearch for more papers by this authorJ. F. RIOU, J. F. RIOU Anticancer Research Program, Centre de Recherche Rhone-Poulenc Rorer, Vitry F-94403, FranceSearch for more papers by this authorS. PARODI, S. PARODI Laboratory of Experimental Oncology, National Cancer Institute and Department of Clinical and Experimental Oncology, University of Genoa, Genoa I-16132, ItalySearch for more papers by this authorP. M. O'CONNOR, P. M. O'CONNOR Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USASearch for more papers by this author P. RUSSO, Corresponding Author P. RUSSO Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA Laboratory of Experimental Oncology, National Cancer Institute and Department of Clinical and Experimental Oncology, University of Genoa, Genoa I-16132, Italy To whom correspondence should be addressed: Department of Experimental Oncology, National Cancer Institute, Largo Rosanna Benzi 10, I-16132 Genoa, Italy. Phone, +39 010 5600212; fax, +39 010 5600210. e-mail, [email protected]Search for more papers by this authorC. OTTOBONI, C. OTTOBONI Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA Laboratory of Experimental Oncology, National Cancer Institute and Department of Clinical and Experimental Oncology, University of Genoa, Genoa I-16132, ItalySearch for more papers by this authorC. FALUGI, C. FALUGI Department of Developing Biology, University of Genoa, Genoa, ItalySearch for more papers by this authorW. REINHOLD, W. REINHOLD Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USASearch for more papers by this authorJ. F. RIOU, J. F. RIOU Anticancer Research Program, Centre de Recherche Rhone-Poulenc Rorer, Vitry F-94403, FranceSearch for more papers by this authorS. PARODI, S. PARODI Laboratory of Experimental Oncology, National Cancer Institute and Department of Clinical and Experimental Oncology, University of Genoa, Genoa I-16132, ItalySearch for more papers by this authorP. M. O'CONNOR, P. M. O'CONNOR Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USASearch for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb09429.xCitations: 4 Oncology Research Division, Agouron Pharmaceuticals, Inc., San Diego, CA 92121, USA Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume886, Issue1ANTICANCER MOLECULES: STRUCTURE, FUNCTION, AND DESIGNDecember 1999Pages 252-256 RelatedInformation

Altered expression and activity of G1/S cyclins and cyclin-dependent kinases characterize squamous cell carcinomas of the head and neck.

Progressive deregulation of the cell-division cycle is thought to contribute to the establishment and progression of neoplasia. Previously, we have documented the in vivo inactivation of p16INK4A, an inhibitor of G1 cyclin-dependent kinases, in squamous cell carcinomas of the head and neck region. In the present study, we extend these findings by examining the expression and functional activity of cyclin-dependent kinases (CDKs) and their regulatory subunits using a model system of cell lines derived from squamous cell carcinomas. Increased activity of CDK4 and 6 was universal in tumor cells compared with normal keratinocytes, reflecting over-expression of either or both kinases. In contrast to other studies, over-expression of cyclin D1, a regulatory subunit of CDK4 and 6, was not observed. Increased activity of CDK2 was less frequent and was related to over-expression of cyclin A and/or E. All tumor cell lines showed increased expression of proliferating cell nuclear antigen compared to normal keratinocytes. Four SCC cell lines, including one tumor-metastasis pair derived from a single patient, failed to express the p15INK4B transcript. Western blot analysis of cell lysates revealed normal or reduced levels of p27KIP1 in tumor cells compared to normal keratinocytes. However, failure to express wild-type p53 was not reflected by lower levels of p21WAF1. Our data suggest that cell-cycle deregulation is likely to occur by multiple mechanisms during the genesis of head and neck squamous cell carcinomas. Furthermore, p16INK4A is likely to be the primary target for inactivation on chromosome 9p21 in these tumors as p15INK4B loss occurs less frequently.

Characteristics of Actinobacillus actinomycetemcomitans invasion of and adhesion to cultured epithelial cells.

Actinobacillus actinomycetemcomitans (A.a.) is highly implicated in periodontitis. We have developed several in vitro models using the KB oral cell line to examine A.a.-epithelial cell interactions. In support of the use of KB cell line model systems is our finding that A.a. invaded KB and primary gingival cells to the same extent. Invasion is an active event which requires new protein synthesis by both KB and A.a. Like many other intracellular parasites, A.a. invade by receptor-mediated endocytosis. We observed that internalized A.a. were surrounded by foci of actin which had been transported from the periphery of the KB cell. Adhesion of A.a. to KB cells occurred rapidly and stimulated the formation of microvilli. Adhesion is affected by both host factors (saliva, serum, [NaCl]) and culture conditions. Multiple determinants [fimbriae, outer membrane proteins, vesicles, and/or an extracellular amorphous material (ExAmMat)] which are either associated with the A.a. surface or are released into the milieu are involved. We determined that ExAmMat can convey adhesiveness to weakly adherent A.a. and to at least one other oral species (Streptococcus parasanguis).

Tumor necrosis factor-alpha downregulates protein S secretion in human microvascular and umbilical vein endothelial cells but not in the HepG- 2 hepatoma cell line

Protein S deficiency, which is associated with thrombosis, can either be inherited or acquired. Recently, we reported that a decrease in free protein S was observed in 19 of 25 persons with HIV/AIDS. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been reported to be elevated in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients and has been shown to induce a procoagulant state on the surface of endothelial cells. We report here that recombinant TNF-alpha (rTNF-alpha) downregulated protein S synthesis in the SV-40T transfected human microvascular endothelial cell line (HMEC-1) model system by approximately 70% and in primary human umbilical vein and dermal microvascular endothelial cell cultures by approximately 50%. Using the HMEC-1 model, Northern blot analysis showed a decrease in protein S RNA at 24 hours that was corroborated by Western blot analysis and enzyme- linked immunosorbent assay (ELISA) quantification. Evidence supporting the specificity of the TNF-alpha effect included the following: (1) TNF- alpha down-regulation of protein S was completely blocked by TNF neutralizing antibody; (2) the effect was transient, and protein S was restored to near normal levels after TNF was removed from cell cultures; (3) an antibody directed to the TNF RI (55-kD receptor) was shown to mimic the action of TNF-alpha on HMEC-1 cells; and (4) other proinflammatory cytokines, interleukin (IL)-1, IL-6, and TGF-beta, had no effect on protein S secretion. However, TNF-alpha showed no regulatory control over protein S synthesis in the human hepatocellular carcinoma cell line HepG-2. We suggest that TNF-alpha downregulation of protein S may be a mechanism for localized procoagulant activity and thrombosis recently reported in some AIDS patients with associated protein S deficiency.

Tumor Necrosis Factor-a Downregulates Protein S Secretion in Human Microvascular and Umbilical Vein Endothelial Cells But Not in the HepG-2 Hepatoma Cell Line

Protein S deficiency, which is associated with thrombosis, can either be inherited or acquired. Recently, we reported that a decrease in free protein S was observed in 19 of 25 persons with HIV/AIDS. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been reported to be elevated in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients and has been shown to induce a procoagulant state on the surface of endothelial cells. We report here that recombinant TNF-alpha (rTNF-alpha) downregulated protein S synthesis in the SV-40T transfected human microvascular endothelial cell line (HMEC-1) model system by approximately 70% and in primary human umbilical vein and dermal microvascular endothelial cell cultures by approximately 50%. Using the HMEC-1 model, Northern blot analysis showed a decrease in protein S RNA at 24 hours that was corroborated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) quantification. Evidence supporting the specificity of the TNF-alpha effect included the following: (1) TNF-alpha down-regulation of protein S was completely blocked by TNF neutralizing antibody; (2) the effect was transient, and protein S was restored to near normal levels after TNF was removed from cell cultures; (3) an antibody directed to the TNF RI (55-kD receptor) was shown to mimic the action of TNF-alpha on HMEC-1 cells; and (4) other proinflammatory cytokines, interleukin (IL)-1, IL-6, and TGF-beta, had no effect on protein S secretion. However, TNF-alpha showed no regulatory control over protein S synthesis in the human hepatocellular carcinoma cell line HepG-2. We suggest that TNF-alpha downregulation of protein S may be a mechanism for localized procoagulant activity and thrombosis recently reported in some AIDS patients with associated protein S deficiency.

Characterization of IL-2 receptor expression and function on murine macrophages.

This study was designed to examine the expression and function of IL-2R on murine macrophages. We used a model system of murine macrophage cell lines (ANA-1 and GG2EE) that was established by infecting normal murine bone marrow-derived cells with the J2 (v-raf/v-myc) recombinant murine retrovirus. ANA-1 macrophages did not constitutively express detectable levels of mRNA for the p55, IL-2R alpha. However, a brief exposure to IFN-gamma was sufficient to induce IL-2R alpha mRNA in ANA-1 macrophages. Flow cytometric analysis indicated that ANA-1 macrophages expressed low constitutive levels of IL-2R alpha on their cell surface that were augmented after treatment of the cells with IFN-gamma. Affinity binding and cross-linking of [125I]IL-2 to ANA-1 macrophages demonstrated that IL-2R alpha and the p70-75, IL-2R beta were both present on ANA-1 macrophages constitutively. IFN-gamma increased the expression of IL-2R alpha on ANA-1 macrophages but did not increase the expression of IL-2R beta on these macrophages. Although IL-2 alone did not induce the tumoricidal activity of ANA-1 macrophages, IL-2 acted synergistically with IFN-gamma to induce macrophage tumoricidal activity. These data demonstrate the expression of IL-2R on murine macrophage cell lines and establish the role of IL-2 as a costimulator of macrophage-mediated tumoricidal activity.

In Vitro Assessment of Cytotoxic Agents in Murine Cancers: Comparison Between Antiproliferative and Antimetabolic Assays<xref ref-type="fn" rid="FN2">2</xref>

Different methods were compared for the in vitro evaluation of the therapeutic effects of the antineoplastic agents doxorubicin, cisplatin, fluorouracil, and vinblastine sulfate in a model system of murine tumor cell lines consisting of L1210 leukemia, P815 mast cell leukemia, and B16 melanoma. Excellent correlations were found with the in vivo effects with the use of a soft agar clonogenic assay, irrespective of the method of growth assessment (i.e., visual colony counting or incorporation of tritiated thymidine in proliferating colonies). Drug effects on the proliferation of tumor cell lines in liquid medium frequently led to an overestimation or underestimation of the actual in vivo effects. Direct incorporation of the radiolabeled precursors thymidine, uridine, and leucine after pretreatment with drugs always led to the prediction of resistance and was therefore considered unreliable.

Metastasis-Enhancing Effect of Heparin and Its Relationship to a Lipoprotein Factor<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The effect of low-dose heparin on spontaneous metastasis formation was studied with the PA-III rat prostate adenocarcinoma cell line model system. In LW rats given heparin iv at a dose of 1,000 U/kg body weight (three times/wk), the metastatic spread of implanted PA-III cells from the footpad through ipsilateral lymphatics to the lungs was enhanced. The weights of the draining lymph nodes (popliteal, inguinal, and axillary) and the number of lung tumor colonies were significantly increased compared with those in the saline-treated control tumor-bearing rats. The growth of the primary tumor was also enhanced. Heparin alone did not induce enlarged lymph nodes in rats nor did it change the growth pattern of PA-III cells in vitro. The accelerated metastatic spread of the PA-III cells was possibly related to the destruction of the oncolytic activity of the very low-density lipoprotein by the lipoprotein lipases induced by the iv administration of heparin. However, the possibility that other mechanisms could be operative in this phenomenon has not been ruled out.

Binding of retinoids to human breast cancer cell lines and their effects on cell growth.

Vitamin A and its analogues (retinoids) regulate the differentiation of epithelial tissues. Retinoids inhibit the induction of rat mammary cancers by carcinogens in vivo, and cellular binding proteins for retinoids have been demonstrated in some human breast cancer samples. In this study, we examined the model system of human breast cancer cell lines in long-term tissue culture for effects of retinoids on growth and for the presence of cellular retinoid binding proteins. Retinoic acid and retinol inhibit the growth of of MCF-7, Hs578T, and ZR-75-B cell lines. Retinoic acid is more potent than retinol in this regard: 50% growth inhibition is achieved by 6 nM retinoic acid in ZR-75-B and by 700 nM in MCF-7 and Hs578T, whereas 5-8 muM retinol is required in all three cell lines. The time to onset of growth inhibition varies markedly between cell lines and is not related to cell density or doubling time. Retinoic acid increases the doubling time of MCF-7 and ZR-75-B by two- to threefold, but causes cell death in Hs578T. The growth inhibition is reversible in every cell line by removal of retinoic acid. Specific and distinct binding of [(3)H]retinoic acid and [(3)H]retinol is present in cytosols of MCF-7 and Hs578T cells as assessed by sucrose density gradient centrifugation. In ZR-75-B, [(3)H]retinoic acid binding was present, but no binding of [(3)H]retinol was detectable. This study reveals that retinoids may play an important role in the regulation and treatment of human breast cancer and that human breast cancer cell lines represent a useful model to study this role.