Normal Human Fibroblast Lines Research Articles

Biomarkers and Precision Therapy for Primary Immunodeficiencies: An In Vitro Study Based on Induced Pluripotent Stem Cells From Patients

Ataxia telangiectasia (AT) and Aicardi-Goutières syndrome (AGS) are inherited disorders of immunity with prevalent neurological phenotype. Available treatments are only partially effective, and the prognosis is poor. Induced pluripotent stem cells (iPSCs) are obtained by reprogramming patient somatic cells, preserving the donor individual genetic heritage and creating patient-specific disease models, useful to investigate pathogenesis and drug effects and to develop precision therapies. The aim is to investigate the cytotoxicity of a panel of immunomodulators using iPSCs of patients with AT or different forms of AGS (AGS1, AGS2, and AGS7). iPSCs were obtained by reprogramming AT and AGS patients' cells and, as a control, the BJ normal human fibroblast line, using Sendai virus. Cytotoxic effects of two drugs proposed to treat respectively AT and AGS (dexamethasone and mepacrine) were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 72hours' exposure. Data were obtained also for other immunomodulatory drugs (thioguanine, mercaptopurine, thalidomide, and lenalidomide). Relative expression of genes involved in the tested drug pathways was analyzed. AGS7-derived iPSCs displayed altered viability when treated with a low dose of mepacrine and higher expression of cyclic guanosine monophosphate-adenosine monophosphate synthase, which is the main target for mepacrine action. AGS7-derived iPSCs were also more sensitive to thioguanine, while AGS2 and AT iPSCs were less sensitive to this medication than the BJ-iPSC. All iPSCs were equally sensitive to mercaptopurine and resistant to dexamethasone, thalidomide, and lenalidomide. This work establishes an innovative in vitro model that is useful to investigate the mechanisms of drugs potentially effective in AT and AGS.

Cytotoxic and anticancer effects of the triorganotin compound [(C6H5)3Sn(cmbzt)]: An in vitro, ex vivo and in vivo study

Since the initial success of cisplatin, metal complexes and organometallic compounds have been gaining growing interest in cancer therapy. It is well known that organotin(IV) compounds display strong biological activity. The triorganotin compound [(C6H5)3Sn(cmbzt)] (cmbzt=5-chloro-2-mercaptobenzothiazole) (SnCMB), was tested for its antiproliferative and antitumour activities. Two sets of experimental procedures were followed: (1) In vitro and ex vivo procedures included the study of the cytotoxic activity of the complex against leiomyosarcoma cells (LMS) and on a normal human fibroblast line (MRC5) by the MTT assay (cell proliferation), colony formation efficiency and flow cytometric analysis with Annexin V-FITC. The anticoagulation properties of the complex were also studied. (2) In vivo procedures included acute toxicity studies and finally administration of the complex to tumour bearing Wistar rats. The results showed that the complex exhibited potent cytotoxic activity (LMS IC50=155nM) and induced significant apoptosis against LMS cells. Acute toxicity studies on Wistar rats presented kidney and liver toxicity at a single dose of 40mg/kg body wt. Furthermore, antitumour activity studies on sarcoma bearing Wistar rats revealed that SnCMB complex, administrated in two different therapeutic schemes (treated with 4×2mg/kg body wt every 5days and 3×2.67mg/kg body wt every 10days of SnCMB complex), prolonged mean survival time (by 50% and 70% respectively), but failed to decrease the mean tumour growth rate (MTGR) compared to the control group (p<0.01). In conclusion, the organic complex SnCMB possess potent cytotoxic and antimetastatic effects, and low toxicity introducing it as possible successor of organometallic compounds used nowadays in chemotherapy.

A Computational Model of Cellular Response to Modulated Radiation Fields

To develop a model to describe the response of cell populations to spatially modulated radiation exposures of relevance to advanced radiotherapies. A Monte Carlo model of cellular radiation response was developed. This model incorporated damage from both direct radiation and intercellular communication including bystander signaling. The predictions of this model were compared to previously measured survival curves for a normal human fibroblast line (AGO1522) and prostate tumor cells (DU145) exposed to spatially modulated fields. The model was found to be able to accurately reproduce cell survival both in populations which were directly exposed to radiation and those which were outside the primary treatment field. The model predicts that the bystander effect makes a significant contribution to cell killing even in uniformly irradiated cells. The bystander effect contribution varies strongly with dose, falling from a high of 80% at low doses to 25% and 50% at 4 Gy for AGO1522 and DU145 cells, respectively. This was verified using the inducible nitric oxide synthase inhibitor aminoguanidine to inhibit the bystander effect in cells exposed to different doses, which showed significantly larger reductions in cell killing at lower doses. The model presented in this work accurately reproduces cell survival following modulated radiation exposures, both in and out of the primary treatment field, by incorporating a bystander component. In addition, the model suggests that the bystander effect is responsible for a significant portion of cell killing in uniformly irradiated cells, 50% and 70% at doses of 2Gy in AGO1522 and DU145 cells, respectively. This description is a significant departure from accepted radiobiological models and may have a significant impact on optimization of treatment planning approaches if proven to be applicable invivo.

Abstract 1042: Diminished caspase 8 and c-Myc protein expression in human fibroblasts provides insight into normal cell resistance to TRAIL-mediated apoptosis

Abstract TNF-related apoptosis-inducing ligand (TRAIL)-mediated cell death is currently being exploited in clinical trials for cancer therapy. However, the molecular basis of normal cell resistance to TRAIL-mediated apoptosis, and thus therapeutic index, remains largely unexplored. We found that normal HFF and WI38 fibroblasts expressed slightly lower levels of TRAIL receptor DR5 as compared to human lung and colon cancer cells. While normal and cancer cells showed comparable DcR2 expression, the WI38 and HFF expressed little DR4 and DcR1. Normal fibroblasts were examined for increased expression of proteins known to inhibit TRAIL-mediated cell death. Anti-apoptotic Bcl-xL and Mcl-1 expression in WI38 and HFF was comparable to expression found in cancer cells, while no XIAP expression was detected in the normal fibroblasts. c-Myc, a major determinant of TRAIL sensitivity in cancer cells, was found to be decreased in the normal fibroblasts and thus may be a contributing factor to their escape from TRAIL-induced cell death. NF-κB activation was assessed after 4 hours of TRAIL exposure, but minimal IκB degradation was observed in the normal fibroblasts, suggesting their resistance to TRAIL may not be mediated through induction of pro-survival factors by NFkB. Expression and activation of initiator caspases, required for complete TRAIL signaling, was investigated and although its levels were elevated, no caspase 10 activation was observed in normal WI38 and HFF cells after TRAIL treatment. Extremely low caspase 8 protein expression was detected in the normal human fibroblast lines, at levels barely detectable as compared to tumor cells that generally show robust caspase 8 expression. Reduced caspase 8 expression may be considered as another contributing factor to the resistance of normal cells to TRAIL. Expression and activation of effector caspase 3 was also examined. After TRAIL stimulation, expression of full length caspase 3 declined in normal and tumor cells. However, cleavage of caspase 3 substrate, PARP, was only seen in cancer cells. Our current studies are focused on further analysis of mediators of cell death signaling by TRAIL, whether treatment of normal fibroblasts with proteasome inhibitors, histone deacetylase inhibitors, and/or DNA methylation inhibitors might result in increased caspase 8 protein expression and susceptibility to TRAIL-mediated apoptosis. We are further investigating the effects of serum and growth factors to drive cell proliferation of normal cells on c-Myc expression and TRAIL sensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1042.

Design, synthesis and evaluation of 4,5-di-substituted acridone ligands with high G-quadruplex affinity and selectivity, together with low toxicity to normal cells

A series of 4,5-di-substituted acridones have been designed and synthesized. Several compounds show high affinity for telomeric G-quadruplex DNA in classical and competition FRET assays, together with low duplex DNA affinity, although they do not show activity in a telomerase assay or evidence of telomere shortening. They have low toxicity against a panel of cancer cell lines and a normal human fibroblast line, and produce potent senescence-based long-term growth arrest in the MCF7 and A549 cancer cell lines.

Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

Idiopathic interstitial pneumonias (IIPs) are a collection of pulmonary fibrotic diseases of unknown etiopathogenesis. CC chemokine receptor 7 (CCR7) is expressed in IIP biopsies and primary fibroblast lines, but its role in pulmonary fibrosis was not previously examined. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, 1.0 x 10(6) primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, or histologically normal biopsies were injected intravenously into C.B-17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblast injection, patchy interstitial fibrosis and increased hydroxyproline were present in the lungs of immunodeficient mice. Adoptively transferred nonspecific interstitial pneumonia fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in C.B-17SCID/bg mice. Systemic therapeutic immunoneutralization of either human CCR7 or CC ligand 21, its ligand, significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of IIP fibroblasts. Thus, the present study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CC ligand 21 and CCR7.

Sp1 regulates expression of MET, and ribozyme-induced down-regulation of MET in fibrosarcoma-derived human cells reduces or eliminates their tumorigenicity

Changes in expression of hepatocyte growth factor (HGF) and its receptor, MET, are associated with formation and malignant progression of human tumors. In the present study, 10 of 11 human fibrosarcoma cell lines tested expressed significantly higher levels of MET than were found in a series of normal human fibroblast lines. Still more significant, MET was constitutively phosphorylated in all 11 fibrosarcoma lines, whereas the normal fibroblasts exhibited very low levels of the phosphorylated form. All the cell lines expressed HGF mRNA. To determine the role of MET and/or HGF in tumorigenesis, a fibrosarcoma line expressing high levels of MET protein and low levels of HGF/NK2 mRNA was stably transfected with a hammerhead ribozyme targeting MET. In addition, a fibrosarcoma line expressing high levels of both MET protein and HGF/NK2 mRNA was transfected with a ribozyme targeting MET, or with a ribozyme targeting MET and another targeting HGF. The transfectant cell lines no longer formed tumors, or did so at a greatly reduced frequency and/or longer latency. Because Sp1 is a transcription factor for MET, we assayed the cell lines for their level of Sp1 protein. Sp1 was markedly overexpressed in 7 of the 11 fibrosarcoma lines compared to normal fibroblast lines. Deletion analysis and site-directed mutagenesis of the MET promoter revealed that tandem Sp1 sites in the proximal promoter are critical for transcription of MET. Increased expression of Sp1 in a normal human fibroblast line containing a MET promoter-luciferase construct resulted in a dose-dependent increase in luciferase. Conversely, inhibition of Sp1 binding to DNA in a fibrosarcoma cell line, using an Sp1 decoy, dramatically reduced MET expression. Taken together, these results indicate that in human fibrosarcoma cells, high levels of the phosphorylated form of MET are required for tumor formation and that Sp1 can function to control the level of MET.

Relationship between cellular radiosensitivity and non-repaired double-strand breaks studied for different growth states, dose rates and plating conditions in a normal human fibroblast line

Purpose : The aim of this study was to test under which conditions non-repaired DNA double-strand breaks (dsb) could be used as an indicator of cellular radiosensitivity of normal human fibroblasts. Materials and methods : The experiments were performed with a primary normal skin fibroblast line (NFHH) derived from a healthy donor. Cells were X-irradiated either in exponential or confluent state with high (4 Gy/min) or low dose rate (0.04Gy/min) and either plated immediately or delayed after irradiation. The fraction of clonogenic cells was determined after doses up to 12 Gy using colony forming assay and the number of non-repaired dsb were measured 24h after X-irradiation with doses up to 180 Gy using constant-field gel electrophoresis. Results : Cellular radiosensitivity of NFHH cells was found to depend on all three conditions tested. In contrast, the number of non-repaired dsb was found to depend on dose rate and growth state only. There were, however, no differences for the plating conditions tested. This result was attributed to the almost complete inhibition of cell-cycle progression when cells were plated immediately after irradiation. For the two dose rates and growth conditions, differences in non-repaired dsb were found to correspond with the respective differences measured for the cellular radiosensitivity, and these data agreed fairly well with the correlation previously found for 11 fibroblast lines varying in dsb repair capacity. Conclusions : For irradiation followed by delayed plating only, non-repaired dsb can be used to predict the cellular radiosensitivity.

Ku70/80 gene expression and DNA-dependent protein kinase (DNA-PK) activity do not correlate with double-strand break (dsb) repair capacity and cellular radiosensitivity in normal human fibroblasts.

The expression of the Ku70 and Ku80 genes as well as the activity of the DNA-dependent protein kinase (DNA-PK) were studied in 11 normal human fibroblast lines. The proteins studied are known to be part of a double-strand break (dsb) repair complex involved in non-homologous recombination, as was demonstrated for the radiosensitive rodent mutant cell lines of the complementation groups 5–7. The 11 fibroblast lines used in this study represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy, SF3.5 Gy, ranging from 0.03 to 0.28. These differences in cell survival were previously shown to correlate with the number of non-repaired dsbs. We found that the mRNA signal intensities of both Ku70 and Ku80 genes were fairly similar for the 11 cell lines investigated. In addition, the DNA-PK activity determined by the pulldown assay was fairly constant in these fibroblast lines. Despite the correlation between cell survival and dsb repair capacity, there was no correlation between dsb repair capacity and DNA-PK activity in the tested normal human fibroblast lines. Obviously, in this respect, other proteins/pathways appear to be more relevant. © 1999 Cancer Research Campaign

Dose-rate effects between 0.3 and 30 GY/H in a normal and a malignant human cell line

Purpose : This study used continuous “intermediate” dose rate irradiation (0.3–30 Gy/h) to compare the capacity for and repair of sublethal radiation damage in different cell lines growing in tissue culture. Methods and Materials : Two human cell lines were studied; one was derived from normal human fibroblasts (AG1522) and the other from a squamous cell carcinoma of the uterine cervix (HTB-35). Dose-response curves for clonogenic survival were determined following irradiation of plateau-phase cultures at five different dose rates: 22.6, 6.12, 3.65, 1.04, and 0.38 Gy/h. Subculture following irradiation was delayed for 8–24 to allow for the full repair of “potentially lethal damage.” Results : A significant dose-rate effect was seen in both cell lines. For irradiation at the highest dose rate, survival at 2 Gy (SF2) and the α β ratio were similar for the two cell lines (approximately 0.7 and 8.0 Gy, respectively) but the half-time of repair of sublethal damage was estimated to be approximately five times longer in the normal human fibroblast line (154 min) than in the carcinoma (31 min) cell line. Conclusion : These results indicate that measuring the dose-rate effect between 0.3 and 30 Gy/h is a useful way to identify and quantify differences in sublethal damage repair between cell lines. To the extent that in vitro and in vivo repair parameters are similar, and that representative tumor biopsy specimens can be examined in this way, this approach may provide a prospective way of determining the dose rate (brachytherapy) or fractionation schedule that will optimize the therapeutic ratio.

Comparison of Thermoradiosensitization in Two Human Melanoma Cell Lines and One Fibroblast Cell Line by Concurrent Mild Hyperthermia and Low-Dose-Rate Irradiation

Two human melanoma cell lines, one radioresistant (SK-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD.

The use of asymmetric-field inversion gel electrophoresis to predict tumor cell radiosensitivity

The success of a predictive assay for radiotherapy relies on the use of one or more tumor cell traits that equate with tumor radioresistance or radio sensitivity. These traits can be divided into intrinsic (genetic) and extrinsic (epi-genetic) factors. Most probably, a tumor's response to radiotherapy will be influenced by both of these sets of traits. Radiobiological analysis of cultured cells derived from explanted tumors of head and neck patients has shown that in vitro survival of tumor cells is not the only factor affecting tumor radiocurability. Two possible reasons are the high degree of selection involved in growing the cells in vitro and the inability to assess the contribution of the cell-cell contact effect with cultured cells. A possible means of overcoming both of these problems would be an assessment of the radiosensitivity of the cell population immediately after removal from the tumor. Since a good correlation exists between intrinsic cellular radioresistance and DNA double-strand break repair (DSBR) as assayed by the Neutral Elution technique [21), we have investigated the feasibility of using asymmetric field inversion gel electrophoresis (AFIGE) in identifying resistant tumor cells in vitro. AFIGE has several advantages over neutral elution in that it is faster (approximately 60–80 samples can be run on the same agarose gel) and, most importantly, one can visualize DNA damage and repair by staining the DNA with ethidium bromide: Analysis of five squamous cell carcinoma cell lines derived from head and neck cancers and one normal human fibroblast line showed a significant correlation ( r 2 = 0.92, p ⩽ 0.006) between their extent of DSBR after one hour of repair following 10 Gy of ionizing radiation and their radiosensitivity ( D 0, SF 2Gy). This correlation between DSBR kinetics and radiosensitivity was independent of dose, and there was no correlation between radiosensitivity and damage induction. Thus, AFIGE may overcome some of the problems of clonogenic assays in determining tumor radio sensitivity, particularly if combined with a technique to determine the oxygen profile of individual tumors.

Newcastle disease virus selectively kills human tumor cells

Newcastle disease virus (NDV), strain 73-T, has previously been shown to be cytolytic to mouse tumor cells. In this study, we have evaluated the ability of NDV to replicate in and kill human tumor cells in culture and in athymic mice. Plaque assays were used to determine the cytolytic activity of NDV on six human tumor cell lines, fibrosarcoma (HT1080), osteosarcoma (KHOS), cervical carcinoma (KB8-5-11), bladder carcinoma (HCV29T), neuroblastoma (IMR32), and Wilm's tumor (G104), and on nine different normal human fibroblast lines. NDV formed plaques on all tumor cells tested as well as on chick embryo cells (CEC), the native host for NDV. Plaques did not form on any of the normal fibroblast lines. To detect NDV replication, virus yield assays were performed which measured virus particles in infected cell culture supernatants. Virus yield increased 10,000-fold within 24 hr in tumor and CEC supernatants. Titers remained near zero in normal fibroblast supernatants. In vivo tumoricidal activity was evaluated in athymic nude Balb-c mice by subcutaneous injection of 9 × 10 6 tumor cells followed by intralesional injection of either live or heat-killed NDV (1.0 × 10 6 plaque forming units [PFU]), or medium. After live NDV treatment, tumor regression occurred in 10 out of 11 mice bearing KB8-5-11 tumors, 8 out of 8 with HT-1080 tumors, and 6 out of 7 with IMR-32 tumors. After treatment with heat-killed NDV no regression occurred ( P < 0.01, Fisher's exact test). Nontumor-bearing mice injected with 1.0 × 10 8 PFU of NDV remained healthy. These results indicate that NDV efficiently and selectively replicates in and kills tumor cells, but not normal cells, and that intralesional NDV causes complete tumor regression in athymic mice with a high therapeutic index.

A novel secretory tumor necrosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to the adhesion receptor CD44.

TSG-6 cDNA was isolated by differential screening of a lambda cDNA library prepared from tumor necrosis factor (TNF)-treated human diploid FS-4 fibroblasts. We show that TSG-6 mRNA was not detectable in untreated cells, but became readily induced by TNF in normal human fibroblast lines and in peripheral blood mononuclear cells. In contrast, TSG-6 mRNA was undetectable in either control or TNF-treated human vascular endothelial cells and a variety of tumor-derived or virus-transformed cell lines. The sequence of full-length TSG-6 cDNA revealed one major open reading frame predicting a polypeptide of 277 amino acids, including a typical cleavable signal peptide. The NH2-terminal half of the predicted TSG-6 protein sequence shows a significant homology with a region implicated in hyaluronate binding, present in cartilage link protein, proteoglycan core proteins, and the adhesion receptor CD44. The most extensive sequence homology exists between the predicted TSG-6 protein and CD44. Western blot analysis with an antiserum raised against a TSG-6 fusion protein detected a 39-kD glycoprotein in the supernatants of TNF-treated FS-4 cells and of cells transfected with TSG-6 cDNA. Binding of the TSG-6 protein to hyaluronate was demonstrated by coprecipitation. Our data indicate that the inflammatory cytokine (TNF or IL-1)-inducible, secretory TSG-6 protein is a novel member of the family of hyaluronate binding proteins, possibly involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis.

The Effects of Hyperthermia and Ionizing Radiation in Normal and Ataxia Telangiectasia Human Fibroblast Lines

The effects of 45 degrees C hyperthermia and gamma radiation have been studied in three normal human fibroblast lines (GM38, GM730, WI38) and compared to the effects in two lines derived from patients with the hereditary disease ataxia telangiectasia (AT3BI, AT5BI). All lines, both normal and gamma-sensitive AT, showed a similar resistance to killing by heat alone, suggesting that the defect responsible for the increased radiation sensitivity in AT lines does not confer increased heat sensitivity. Shouldered survival curves were obtained in each case indicating the ability to accumulate sublethal heat damage. All normal and AT cell lines exhibited increased resistance to the lethal effects of heat in response to a thermal stress, indicating that the defect that causes radiosensitivity in AT cell lines does not prevent the induction of thermotolerance. Heat (45 degrees C, 30 min) was shown to increase the sensitivity of the normal cell lines to killing by gamma radiation. The thermal enhancement ratios obtained ranged from about 2.5 to 3.0. The same heat treatment, however, produced very little increase in the radiation sensitivity of the AT cells. Thermal enhancement ratios of about 1.2 were obtained in these lines. We hypothesize that, in normal cells, this heat treatment inactivates the process which is already defective in AT lines, and that this process may be required for the proper rejoining of double-strand breaks produced during the repair of other radiation-induced lesions.

Cell surface-associated growth inhibitory proteins: Evidence for conservation between mouse and human cell lines

The ability of the normal human fibroblast line (IMR91) to exhibit density-dependent regulation of growth has been examined. The line exhibits density-dependent regulation of growth; saturation density in 15% fetal bovine serum is 2 × 10 5 cells/cm 2. Membranes prepared from confluent monolayers of these cells contained growth inhibitory factors to both exponentially growing IMR91 and Swiss 3T3 cells. This factor(s) appears to be similar to a previously described factor found on the surface of Swiss 3T3 cells [14]. The inhibition of DNA synthesis in growing IMR91 cultures by membranes was both time- and concentration-dependent. The effect was reversible by high serum. Specificity experiments utilizing membranes prepared from Swiss 3T3 cells indicated some species specificity for inhibition by membranes, but this specificity was no longer exhibited by solubilized membrane preparations. These results are compatible with the suggestion that both the growth inhibitory factors and their receptors are conserved through evolution.

High-affinity [3H]choline accumulation in cultured human skin fibroblasts.

[3H]Choline can be transported across cell membranes by high-affinity (KT less than 5 microM) and low-affinity (KT much greater than 5 microM) systems. High-affinity choline accumulation (HACA) has been demonstrated in synaptosomes made from cholinergic brain regions such as the hippocampus and caudate-putamen. In cell culture, HACA has been demonstrated in glia and avian telencephalon, dissociated spinal cord, and muscle fibroblasts. We examined [3H]choline accumulation in a single normal human fibroblast line cultured from skin biopsy. [3H]Choline accumulation was temperature-dependent and linear with incubation time up to 6 min at 0.125 microM-choline. The apparent KT for [3H]choline was 5 microM, which is similar to that observed in avian fibroblasts. Isoosmotic replacement of Na+ with either Li+ (144 mM) or sucrose (288 mM) severely reduced [3H]choline accumulation (by 70-90%). Pre-incubation with ouabain (100 microM), sodium orthovanadate (100 microM), or 2,4-dinitrophenol (100 microM), or replacement of Ca2+ by Mg2+ had little or no effect on subsequent [3H]choline accumulation. [3H]Choline accumulation was inhibited by hemicholinium-3 (HC-3); after pre-incubation in HC-3 at 37 degrees C for 10 min, the IC50 (at 0.125 microM-choline) was 5.6 microM. The HC-3 sensitivity, Na+ dependence, and low KT suggest that human skin fibroblasts have a high-affinity transport system for choline.

In vitro approach to a correlation of cell susceptibility to viral infection with HL-A genotypes and other biological markers. 1.

SummaryIn vitro tests of the susceptibility of 30 normal human fibroblast lines to infection by herpes hominis, adenovirus-7, echovirus-11 and measles viruses have shown significant variations in individual host susceptibility to infection by one or more of these agents. Resistance of an individual cell line to a given virus appears to be specific for that virus, and is not necessarily associated with resistance of the same cells to other viruses.Family studies utilizing fibroblasts obtained from parents and from siblings geno-typed for the HL-A system and other biological markers suggest that, if susceptibility to viral infection in man is determined by a host cell genome, this type of genetic control may be of a multigenic nature. No pattern of dominant inheritance has been observed in the limited studies reported here. The results suggest, however, the possibility that the host's HL-A genotype or genes closely linked to the HL-A chromosomal region may play a role in conditioning resistance to viral inf...