VA13 Cells Research Articles

Differential cytotoxicity and DNA-damaging effects produced in human cells of the Mer+ and Mer- phenotypes by a series of alkyltriazenylimidazoles.

A series of alkyltriazenylimidazoles have been investigated for their differential cytotoxicity towards the HT-29 (Mer+) and BE (Mer-) cell lines and for their ability to cause DNA strand breaks and cross-links. A monomethyltriazene, and some hydroxymethyltriazene derivatives capable of generating the monomethyltriazene in situ, were preferentially cytotoxic towards the BE cell line compared with the HT-29 cell line, with very close similarity in the differential toxicity to the analogous monochloroethyltriazene. In contrast, the dimethyl- and monoethyltriazenes in the series display reduced toxicity towards the BE cell line with little or no differential toxicity between BE and HT-29 cell lines. With another pair of human cell lines, the IMR-90 (Mer+) and VA-13 (Mer-) cells, the monomethyl- and monochloroethyltriazenes were again more cytotoxic to the Mer- cells. Neither the formation of DNA single-strand breaks or DNA-protein cross-links could account for the differential cytotoxicity observed in the Mer+ and Mer- cells. More importantly, the inability of the monofunctional monomethyltriazene to cross-link DNA tends to question the role of DNA inter-strand cross-linking as a mechanism for cell killing by chloroethylating agents.

Recombinant human tumor necrosis factor — I. Cytotoxic activity in vitro

Cytotoxic activity of recombinant human TNF (rHu-TNF) on various human cell lines was examined in vitro. rHu-TNF exerted a cytostatic effect on various types of human tumor cells such as carcinoma, sarcoma, leukemia, melanoma and other types. When the cytocidal effect was examined on the tumor cells which were cytostatically susceptible to rHu-TNF, the cytocidal effect of rHu-TNF was also noticed on many of these tumor cells. However, some tumor cells were affected cytostatically only. Human diploid cells were not affected cytostatically or cytocidally by rHu-TNF. WI-38 VA13 cells which are an SV-40-transformed derivative of WI-38 diploid cells, were affected both cytostatically and cytocidally by rHu-TNF. These results suggest that rHu-TNF exerts cytostatic and cytocidal effects against a broad spectrum of human tumor cells, and its cytotoxic activity is tumor-specific.

Postreplication repair in ultraviolet-irradiated human fibroblasts: formation and repair of DNA double-strand breaks.

A neutral filter elution assay was used to determine if the postreplicational formation and repair of DNA double-strand breaks (DSB) occurs in u.v.-irradiated human cells. Excision-deficient XP12 cells were pulse-labeled with [3H]thymidine after u.v. irradiation (1.5-3 J/m2), and the nascent DNA was followed during repair incubation. With increasing u.v. radiation fluences, an increasing fraction of DNA was eluted at a fast rate, indicating that DSB were produced. The maximum yield of DSB was observed after about 24 h of postirradiation incubation at 37 degrees C. Similar results were also obtained with repair-proficient VA13 cells when irradiated at much higher fluences (7.5-15 J/m2). It is concluded that, at the u.v. radiation fluences used in this work, the DSB produced in u.v.-irradiated human cells are the result of post-replication repair events, and at incubation times greater than 24 h some of these DSB are repaired.

Inhibition of tumor cell growth by protein factors derived from the developing mouse brain.

It was recently reported (Endoh et al. 1981, Exp Cell Biol 49:272-277) that conditioned medium of neonatal mouse brain (CM-NB) inhibited the growth of mouse neuroblastoma cells. In this work we fractionated CM-NB by size exclusion high performance liquid chromatography, and separated two active principles (28,000 and 62,000 daltons) Each or a combination of the 28,000 and 62,000 dalton fractions showed a differential inhibitory effect on DNA synthesis or clonal growth of the three human lung cell lines: the normal diploid fibroblast WI38 cells were less susceptible than their simian virus 40-transformed VA13 cells and carcinoma A549 cells. This preferential growth-inhibition of malignant cells was also observed for rat fibroblast 3Y1 and its simian virus 40-transformed W3Y cells, and for two other normal and five other malignant cell lines. The growth-inhibitory activity of CM-NB or the 28,000 and 62,000 dalton fractions was lost by pronase, trypsin, tetrahydrofuran, acetonitrile, or dithiothreitol in the presence of guanidine, and also labile to heat, vigorous agitation, or freeze-thawing. The activity was also found in the conditioned medium of prenatal mouse brain, but not in either the conditioned medium of the adult brain and of the secondary culture of the neonatal brain, or in the homogenate and rinsing fluid of the neonatal brain. Thus the mouse brain at the terminal stage of ontogenesis liberates proteinaceous factors, which exhibit a preferential growth-inhibition of tumor or transformed cells and act on malignant cells of human and rodent origin.

Pneumocystis carinii: Growth variables and estimates in the A549 and WI-38 VA13 human cell lines

Recent studies indicate that rat Pneumocystis carinii can be propagated in the A549 cell line, an alveolar epithelioid cell line derived from human lung carcinoma. In the present study, growth of P. carinii was compared in the A549 cell line and the WI-38 VA13 subline 2RA, an SV40 transformed derivative of the human fetal fibroblast cell line with epithelioid morphology. Similar P. carinii growth occurred in both cell lines under optimal conditions, but the WI-38 VA13 cell line was usually more sensitive to changes in the culture system. Growth of P. carinii was affected by temperature, environmental gas mixture, motion of the cultures, and source and concentration of serum additives, but not by the presence of antibodies in the medium. A technique was developed for quantitating P. carinii in the lung inoculum which permitted analysis of P. carinii growth during the first 24 hr of culture. Inverted microscope and oil immersion phase-contrast microscopy were very helpful in monitoring the organism's stages of development and viability. Thus, this culture system should be helpful in establishing standard methodology for in vitro work with P. carinii.

Acidic glycosaminoglycans of SV40-transformed Werner's syndrome cells.

Acidic glycosaminoglycans (AGAGs) were isolated from the culture fluids of a SV40-transformed permanent line (PSV811) of the Werner syndrome skin cells and of a SV40-transformed permanent line (VA13) of WI-38 normal lung cells. The relative composition of component AGAGs was estimated by acetate membrane electrophoresis followed by Alcian blue staining and desitometry. Hyaluronic acid (HA) was the major AGAG component in all the preparations tested. The relative content of HA fluctuated considerably among the preparations obtained in independent experiments and among the results using three different buffer systems. However, the proportion of HA was persistently greater in PSV811 culture fluid than in VA13 culture fluid. VA13 cells released an appreciable amount of heparan sulfate as the second major AGAG component, but PSV811 released only a small amount. These results show a difference between the two cell lines, and suggest that PSV811 may possibly reflect the association of the Werner syndrome with abnormal HA metabolism.

The carbohydrate structure of human fibronectins: A comparison between normal embryonic lung fibroblasts WI-38 and the SV40 virus transformed cell line VA13

The carbohydrates of human fibronectin released from non-transformed human fibroblasts WI-38 have been compared with those of fibronectin released from SV40 virus transformed WI-38/VA13 cells and those of fibronectin prepared from human plasma. The majority of the bi-antennary glycopeptides of fibronectin released from WI-38 fibroblasts was not sialylated at the terminal galactosyl residues, but was fucosylated at the coreN-acetylglucosaminyl residue directly linked to a peptide (structure A, below). Most of the minor sialylation detected was linked α2–3 to galactose. In contrast, the majority of the bi-antennary glycopeptides released from the transformed VA13 cells was highly sialylated at the terminal galactosyl residues with both α2–3 and α2–6 linkages, but was only partially fucosylated at the coreN-acetylglucosaminyl residue (structure B, below). This structure was similar to that of the bi-antennary glycopeptide of human plasma fibronectin which was, however, predominantly sialylated with an α2–6 linkage (structure C, below). These human fibronectins, regardless of their source, lack a high molecular weight lactosaminoglycan structure.

Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells.

In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97% by aphidicolin at 10 micrograms/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30% and 90% depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90% at 100 micrograms/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 micrograms/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase alpha and a non-alpha DNA polymerase (possibly DNA polymerase beta), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase alpha in UDS favored DNA synthesis in the intranucleosomal region.

Serological Detection of Common Human Melanoma Membrane Antigens by Microcomplement Fixation and Immunofluorescence

An antiserum to human melanoma antigens was obtained from a melanoma patient after immunization with autologous irradiated cultured melanoma cells and bacillus Calmette-Guerin. Using the microcomplement fixation assay, the antiserum, at a titer of 1/1,800, was noted to bind strongly with 7 of 10 allogeneic cultured human melanoma cells lines. However, using the indirect immunofluorescence test and serum at a much lower titer (1/8), only 3 of the 10 melanoma cell lines were positive. Using both microcomplement fixation and indirect immunofluorescence, no significant reactivity was noted in several nonmelanoma cell lines including Hela, human lung adenocarcinoma, human prostatic carcinoma, WI-38 and VA-13 cell lines. These data suggest that common melanoma membrane antigens exist on 7 of 10 cultured human melanoma cell lines as tested by microcomplement fixation and that this assay is more sensitive than immunofluorescence. These common melanoma membrane antigens may eventually be extracted, purified and used for specific immunodiagnosis and immunotherapy.

Pretreatment of normal human fibroblasts and human colon carcinoma cells with MNNG allows chloroethylnitrosourea to produce DNA interstrand crosslinks not observed in cells treated with chloroethylnitrosourea alone.

Chloroethylnitrosoureas (CNU) are antitumor agents which produce DNA interstrand crosslinks. We have proposed that crosslinks are produced in DNA via monoadduct formation at the guanine-O6 position, followed by a delayed reaction with the opposite DNA strand. Human cells are known to differ in their capacity to repair the O6-methylguanine lesion. One example of this repair capacity is the ability of cells to reactivate adenovirus which has been damaged by in vitro treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cells that repair the virus are designated Mer+ and deficient cells Mer-. In a recent report, we showed a clear correlation between CNU-induced DNA interstrand crosslinking and the Mer phenotype. Mer- cells produced consistently higher levels of interstrand crosslinks than did Mer+ cells. In the present study we have measured the CNU-induced DNA interstrand crosslinking in IMR-90 normal human fibroblasts (Mer+), HT-29 human colon carcinoma cells (Mer+), and VA-13 SV-40 transformed human cells (Mer-) following pretreatment with MNNG. Cells were treated for 1 h with MNNG, then for an additional 1 h with CNU. Comparable levels of CNU-induced DNA interstrand crosslinking were observed in all cell lines. This crosslinking has been previously undetected in the IMR-90 and HT-29 cells. Cytotoxicity studies showed that MNNG pretreatment greatly enhanced the killing of IMR-90 and HT-29 cells by CNU, however, in VA-13 cells the increase in cell kill was smaller. These data suggest that in Mer+ cells a DNA repair system may remove chloroethyl monoadducts before the lethal DNA interstrand crosslinks can form. However, pretreatment of cells with MNNG may saturate this repair system rendering it inoperable.

Transformation-dependent alterations is glycoproteins of extracellular matrix of human fibroblasts. Characterization of GP250 and the collagen-like GP140.

The extracellular matrix, prepared by extraction of confluent cultures of human lung WI-38 fibroblasts with a dipolar tonic detergent, contains four major glycoproteins: fibronectin, GP250, GP170, and GP140. All the glycoproteins can be surface-labeled; however, only fibronectin and GP170 can be readily removed by digestion with trypsin (Carter, W. G., and Hakomori, S. (1981) J. Biol. Chem. 256, 6953-6960). Most of the noncovalently bound GP250, GP170, and GP190, an additional minor glycoprotein, can be dissociated from the matrix by extraction with 8 M urea. The remaining insoluble matrix is stabilized by extensive intermolecular disulfide bonds and contains primarily GP140 and fibronectin (Carter, W. G. (1982) J. Biol. Chem. 257, 3249-3257). Affinity-purified, monospecific antibodies were prepared against GP[140 and fibronectin and utilized for detection of GP140 and fibronectin in extracts and conditioned media of WI-38, WI-38 VA13, WI-26, WI-26 VA4, and HT-1080 cells. Additional affinity-purified, polyspecific antibodies that react with GP250, GP190 GP170, and GP140 were also utilized. Fibronectin, GP250, GP190, GP170, and GP140 were all absent from transformed cells. With the exception of GP140, the absence of these glycoproteins from the matrix of transformed cells was paralleled by their accumulation in the conditioned culture media. Incubation of conditioned culture media with collagenase indicated that GP190, GP170, and GP140, as well as other glycoproteins, were digested. Antibodies to GP140 did not react with any other cellular component indicating that it is not a processing product of other matrix glycoproteins. GP140 has characteristics unlike all reported collagen types and appears to be a new collagen-like glycoprotein. In contrast, neither Gp250 nor fibronectin were sensitive to digestion with collagenase. Antibodies that react with GP250 did not react with fibronectin and vice versa, suggesting that GP250 and fibronectin do not share antigenic determinants. The interaction of labeled fibronectin and the labeled, gelatin-binding domain of fibronectin with cells after fractionation on polyacrylamide gels indicated that GP170 is the primary procollagen receptor for fibronectin in the extracellular matrix. GP140 also bound fibronectin but to a lesser degree. Soluble GP170 and GP190 present in the conditioned medium of cultured cells also bound to insolubilized fronectin, confirming the association of GP170 and GP190 with fibronectin. The interaction of the glycoprotein components in the matrix are discussed in relation to their potential cooperative function in cell attachment and their failure to adhere to the surface of transformed cells.

Possible involvement of DNA polymerases α and β in bleomycin-induced unscheduled DNA synthesis in permeable Hela cells

DNA polymerases involved in bleomycin-induced unscheduled DNA synthesis in some permeable human cells and rodent cells were studied by using selective inhibitors (aphidicolin, 2′,3′-dideoxythymidine-5′-triphosphate and N-ethylmaleimide) for DNA polymerases. The results suggest that both DNA polymerases α and β are involved in bleomycin-induced unscheduled DNA synthesis in permeable HeLa-S3 cells and probably in some other permeable human cells (HEp-2, KB and WI-38 VA-13 cells). Bleomycin-induced unscheduled DNA synthesis in some permeable rodent cells ( SR- C3H He , Balb c 3T3, 3Y1 and XC cells) is mostly attributed to DNA polymerase β.

Visualization of nucleolar substructure in cultured human fibroblasts by magnesium-activated adenosine triphosphatase reaction.

Discrete sites of adenosine triphosphatase (ATPase) activity were demonstrated within the nucleoli of unfixed cultured human fibroblasts (IMR90, VA13, and AG2804 cells) by an adaptation, for electron microscopic cyto-chemistry, of Wachstein and Meisel's lead nitrate method. The majority of nucleoli contained more than one ATPase-positive region, but the total ATPase-positive material appeared to occupy only a minor portion of the nucleolar volume. These regions were roughly spherical with an irregular contour, and at times appeared to be components of perinucleolar chromatin or to be located adjacent to nucleolar interstices. The distribution of these regions within the nucleolus and their segregation by actinomycin D suggested that the ATPase-positive regions correspond to the fibrillar centers, which represent nucleolar organizer regions. The cytochemically demonstrable nucleolar ATPase was strictly dependent on the presence of divalent cations. Optimal reactions was seen at 5 mM Mg2+, but near optimal activity was obtained with lower concentrations of Mg2+ in the presence of Ca2+. Calcium alone and Mn2+ alone produced suboptimal reaction. Studies with different nucleoside phosphates as reaction substrates showed that the enzyme is specific for adenosine derivatives, ATP and dATP being equally good substrates. Guanosine triphosphate, cytidine triphosphate, uridine triphosphate, and d-thymidine triphosphate were ineffective as substrates, as were nucleoside mono- and diphosphates and other phosphate esters tested. It is suggested that the cytochemical ATPase reaction visualized the regions of the nucleolus in which ribosomal DNA of intranucleolar chromatin is undergoing conformational alterations.

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells.

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.

DNA crosslinking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas.

Normal (IMR-90) and simian virus 40-transformed (VA-13) human embryo cells were treated with antitumor nitrosoureas, and the effects on cell viability and cell DNA were compared. All six nitrosoureas tested were more toxic to VA-13 cells than to IMR-90 cells as measured by decrease in cell proliferation or in colony formation. The nitrosoureas capable of generating alkylisocyanates produced a smaller difference between the cell types than did derivatives lacking this capacity. DNA damage was measured by alkaline elution in cells treated with four chloroethylnitrosoureas. Whereas VA-13 cells exhibited dose-dependent interstrand crosslinking, little or none was detected in IMR-90 cells. The IMR-90 cells, however, exhibited at least as much DNA-protein crosslinking as did VA-13 cells. The results can be interpreted in terms of a possible difference in DNA repair between the cell lines.

Binding and degradation of α2-macroglobulin by cultured fibroblasts

We studied the interactions of α 2-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human α 2-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0–4°C, binding was saturated at a concentration of 10–20 μg/ml. At 37°C, radiolabel appered in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated α 2-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated α 2-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified α 2-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less 125I-labeled α 2-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing α 2-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade α 2-macroglobulin. Binding and endocytosis of α 2-macroglobulin by a cell may be a means of modulating proteases in the micro-environment of the cell and during endocytosis.

Adenosine 3′,5′-monophosphate-dependent protein kinase(s) in diploid and SV40 transformed human fibroblasts

Cyclic AMP_dependent protein kinases (EC 2.7.1.37; ATP: protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAE-cellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10–30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activitation induced in the presence of 0.5 M KCl was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 μM prostaglandin E 1 or 1 μM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intracellular cyclic AMP levels were raised by prostaglandin E 1, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.

Repair replication in cultured normal and transformed human fibroblasts

Repair replication in response to ultraviolet irradiation has been studied in normal human diploid fibroblast cultures, WI38, and an SV40 transformant, VA13. Quantitative comparisons have been made using the combined isotopic and density labeling method for assaying repair replication. We find no significant difference in the amount of repair replication performed, its dose response, or the time course between growing and confluent WI38 cells, early passage and senescent cells, or normal WI38 cells and the transformed VA13 cells. When [ 3H]dThd was employed as the isotopic label in the presence of a 30–200 fold excess of unlabelled BrdUrd, apparent differences in repair replication were seen between WI38 cells shortly after subcultivation and cells which had been allowed to reach confluence. These differences were the same over a wide dose range and regardless of the passage number of the cells, but could be influenced by using different serum lots. The differences were not seen, however, when [ 3H]BrdUrd provided the isotopic label; thus they reflect either impurities in the [ 3H]dThd or a slight discrimination by some cellular process.

Protein degradation in human fibroblasts (WI-38). Effects of aging, viral transformation, and amino acid analogs.

Protein degradation occurs more rapidly in senescent WI-38 cultures than in phase II cultures or in SV-40 transformed WI-38 cells (VA-13). The first differences are found in early phase III, when short lived but not long lived proteins are degraded more rapidly. At the end of phase III long lived proteins are also degraded more rapidly as shown by both intermittent perfusion and approach to equilibrium methods. By both methods the rates of protein degradation for the virally transformed derivative are the same as those for phase II WI-38, implying that transformation has not altered these characteristics of protein degradation. WI-38 cells incorporate canavanine, an analog of arginine, into protein. This analog, as well as p-fluorophenylalanine and azetidine carboxylic acid, accelerates the degradation of proteins labeled with [3H]leucine in their presence but does not alter the degradation rates of proteins prelabeled with [14C]leucine in the absence of the analogs. These results imply that the analogs increase the intracellular degradation rates of proteins because they render them more susceptible to the degradative system. Late phase III WI-38 cells may not selectively catabolize proteins containing canavanine as rapidly as do phase II and VA-13 cells. These results imply that the phase III protein degradative system becomes partially defective, thereby losing its ability to rapidly catabolize altered protein which leads to increased levels of abnormal proteins and decreased cell function.