Human Tumor-derived Cell Research Articles

Glutamine as a regulator of DNA and protein biosynthesis in human solid tumor cell lines.

The transport of glutamine by six different human solid tumor-derived cell lines (e.g., breast, colon, liver) was characterized and the impact of glutamine deprivation on rates of tumor cell proliferation and DNA and protein synthesis was assayed. Glutamine is added routinely to cell culture media and its importance for cellular growth has been established. However, carrier-mediated glutamine transport by solid tumors has not been studied extensively, and the mechanisms by which glutamine contributes to cell growth regulation require further investigation. In a panel of different human solid tumor-derived cells, sodium-dependent glutamine transport was characterized in vitro and rates of cell proliferation, protein and DNA synthesis, as well as thymidine transport, were correlated with glutamine concentrations in the culture media. In all cells, regardless of tissue origin, sodium-dependent glutamine transport was mediated almost exclusively by a single carrier. There was a range of Michaelis constants (Km) and maximal transport velocities (Vmax) for the glutamine transporter in each cell type, but the amino acid inhibition profiles were nearly identical, consistent with uptake by the System ASC family of transporters. Rates of cell growth, DNA and protein synthesis, and thymidine transport correlated with the glutamine concentration in the culture media, indicating the central role of this amino acid in regulating cellular proliferation. These data indicate that glutamine transport by all solid tumors is mediated by the System ASC family of transporters. The variation in Km values suggests that some cancers may be better suited to survive in a low glutamine environment than others. The mechanism by which glutamine supports cell proliferation and regulates cell cycle kinetics involves its modulation of DNA and protein biosynthetic rates.

Autocrine Regulation of Membrane Transforming Growth Factor-α Cleavage

Transforming growth factor alpha (TGF-alpha) is biosynthesized as a membrane-bound precursor protein, pro-TGF-alpha, that undergoes sequential endoproteolytic cleavages to release a soluble form of the factor. In the present study, we have analyzed the biosynthesis and regulation of TGF-alpha production in human tumor-derived cell lines that endogenously express pro-TGF-alpha and the epidermal growth factor (EGF) receptor. These cells biosynthesized membrane-anchored forms of the TGF-alpha that accumulated on the cell surface. Membrane-bound pro-TGF-alpha interacted with the EGF receptor, and complexes of receptor and pro-TGF-alpha contained tyrosine-phosphorylated receptor. Activation of the EGF receptor by soluble EGF or TGF-alpha had a dual effect on TGF-alpha production: an increase in pro-TGF-alpha mRNA levels and an increase in pro-TGF-alpha cleavage. These effects were largely prevented by preincubation with an anti-EGF receptor monoclonal antibody that blocked ligand binding. Growth factor autoinduction of cleavage could be stimulated by several second messenger pathways that are activated by the EGF receptor, including protein kinase C and intracellular calcium, and by other alternative mechanisms. EGF-stimulated cleavage of pro-TGF-alpha could be partially blocked by inhibition of these second messenger pathways. These results suggest that juxtacrine stimulation takes place in human tumor cells that coexpress both the EGF receptor and membrane-anchored TGF-alpha and that TGF-alpha is able to induce its own endoproteolytic cleavage by activating the EGF receptor.

Manifestations of chemically induced liver damage

Possible liver damage induced by chemicals or drugs must be detected early during drug development or industrial exposure, although damage is still difficult to predict, especially when immunotoxicity is involved. Liver toxicity may result from cytolysis, steatosis, cholestasis, phospholipidosis, or vascular lesions, most the outcome of a disadvantageous balance between chemicals or metabolites vs protective mechanisms, resulting from chemical dosage, genetic factors, or the immunoallergic status of the patient. Drug metabolism, lipid peroxidation, and thiol oxidation are frequently involved in liver toxicities. Classical guidelines in toxicology propose many methods for liver toxicity assessment: histology; chemical changes in hepatic tissue (lipids, glutathione, enzymes); physiological changes in biosynthesis (proteins, glycoproteins); excretion function (fructose); drug metabolism; and concentrations of related enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase) in blood. In vitro studies in human or animal hepatocytes or tumor-derived cell lines are useful in detecting hepatocellular lesions by cell viability, glutathione concentration, amount of lactate dehydrogenase released, cellular ATP, morphology (blebs), and drug metabolism.

Human astrocytoma cells express a unique chloride current

Human astrocytoma cells were studied using whole-cell patch-clamp recording. Voltage-dependent outwardly-rectifying anion currents were identified in primary cultures of six freshly resected human brain tumors and in seven established anaplastic astrocytoma/glioblastoma cell lines (U251MG, CH235MG, U373MG, U105MG, D54MG, SK-MG-1, and STTG1). Anion currents were not observed in normal, non-neoplastic glial cells, nor in human tumor-derived cells of non-glial origin (melanoma, breast cancer, neuroblastoma, rhabdomyosarcoma). Currents activated at potentials > 50 mV and showed large transients upon termination of voltage steps. Currents reversed at the predicted equilibrium potential for chloride ions and could also be recorded when Cl- was replaced by F-, Br- or I-. Currents were inhibited by the Cl- channel blockers chlorotoxin, DIDS, and DNDS. These Cl- currents may play a role in the growth control of astrocytoma cells.

Membrane topology of the L6 antigen and identification of the protein epitope recognized by the L6 monoclonal antibody.

The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is highly expressed on lung, breast, colon, and ovarian carcinomas and is referred to as the L6 antigen. This antigen is an attractive target for therapeutic intervention due to its high level expression on malignant cells. We have previously reported the isolation of a cDNA encoding the human L6 antigen (H-L6). Here, we report the isolation of a cDNA clone encoding the murine L6 antigen (M-L6). This cDNA contains one long open reading frame, which encodes a 220-amino acid polypeptide that is 78% homologous to H-L6. This protein contains short NH2- and COOH-terminal hydrophilic domains and four hydrophobic regions, each long enough to span the plasma membrane. Each of these hydrophobic domains is separated by a hydrophilic domain, the longest of which contains one possible N-linked glycosylation site and is located between the third and fourth hydrophobic domains. We have previously demonstrated that the murine L6 mAb recognizes a protein epitope expressed on human tumor-derived cell lines. Now, using chimeric cDNA constructs encoding human-murine L6 antigen hybrids in conjunction with monoclonal antibody binding experiments, we show that the 42-residue hydrophilic domain of the L6 antigen, located between the third and fourth hydrophobic domains, is outside the cell and that residues in the NH2-terminal region of this domain are critical for the binding of the murine L6 mAb to H-L6.

Effects of tumor necrosis factor and interferon-β on proliferation and epidermal growth factor binding in young and senescent WI-38 cells

Tumor necrosis factor-alpha (TNF) and various interferons (IFN) have potent cytostatic or cytotoxic effects on a variety of human tumor-derived cell lines. Their effects on normal cells are more controversial. We have examined the effects of TNF and IFN-beta on the proliferation of WI-38 cells in a serum-free, growth factor-supplemented medium and in serum-containing medium. These cells respond to the combination of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dexamethasone by DNA synthesis at a rate and extent equivalent to serum-stimulated cells. TNF has no effect on this growth factor-stimulated proliferation. However, it is stimulatory in serum-containing medium. IFN-beta inhibits DNA synthesis 60 to 70% in both young and senescent cells. TNF and IFN-beta together have a synergistic effect and completely inhibit growth factor-stimulated DNA synthesis in young cells. No synergism was observed with senescent cells. TNF stimulated an increase in the number of EGF specific binding sites two- to threefold in 24 h in both young and senescent cells. This seems to result from a proportional increase in a very high affinity binding site. IFN-beta has little or no effect on EGF binding either alone or in combination with TNF.

Cytochrome P-450 induction in human lung tumor-derived cell lines. Characterisation and effects of inflammatory mediators.

Cytochrome P-450 species (P-450) comprise a polymorphic multigene family of heme-containing enzymes which are essential to the phase-I metabolism of xenobiotics. Induction of P-450 species by drugs and carcinogens has been extensively studied; endogenous regulation of P-450 also occurs during normal development and disease. The aim of this project was to study the in-vitro induction of P-450 and its modulation by inflammatory mediators in the human lung tumor-derived cell lines NCI H322 and NCI H358. The cell lines expressed detectable levels of 7-ethoxyresorufin O-deethylase which could be induced by benzanthracene. After benzanthracene treatment, a protein tentatively identified as isozyme CYP1A1 was detected by Western-blot analysis and a concommitant increase in CYP1A mRNA expression was observed. Optimal induction was observed at a benzanthracene concentration of 5 micrograms/ml with cells grown in RPMI medium containing 10% fetal calf serum. The effects of endotoxin, dexamethasone and five recombinant DNA-derived cytokines, interleukin-1 beta, tumor necrosis factor, and interferons alpha, beta and gamma, on constitutive and benzanthracene-induced ethoxyresorufin O-deethylase activity were examined in NCI H322 cells. Of all the lymphokines studied, only interferon gamma had any marked effect. Administration of this lymphokine strongly suppressed ethoxyresorufin O-deethylase activity in both control and benzanthracene-treated cells.

Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line.

A wild-type p53 gene under control of the metallothionein MT-1 promoter was stably transfected into human colon tumor-derived cell line EB. Repeated inductions of the metallothionein wild-type p53 gene with zinc chloride results in progressive detachment of wild-type p53 cells grown on culture dishes. Examination at both the light and electron microscopic level revealed that cells expressing wild-type p53 developed morphological features of apoptosis. DNA from both attached and detached cells was degraded into a ladder of nucleosomal-sized fragments. Expression of wild-type p53 inhibited colony formation in soft agar and tumor formation in nude mice. Furthermore, established tumors in nude mice underwent regression if wild-type p53 expression was subsequently induced. Regressing tumors showed histological features of apoptosis. Thus, regression of these tumors was the result of apoptosis occurring in vivo. Apoptosis may be a normal part of the terminal differentiation program of colonic epithelial cells. Our results suggest that wild-type p53 could play a critical role in this process.

Expression of phospholipid hydroperoxide glutathione peroxidase in human tumor cell lines

Free radical-induced damage in cells is thought to be involved in the initiation and/or promotion of many cancers. A multilevel antioxidant defense system effective in the protection against free radical damage includes chain-breaking antioxidants and enzymes, such as superoxide dismutase, catalase, and glutathione peroxidases. These latter, glutathione peroxidase (PGX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX), are the only selenoperoxidases present in cells and have been distinct on the basis of mol wt, substrate specificity, and peptide and cDNA fragment sequencing. The ability of PHGPX to reduce phospholipid and cholesterol hydroperoxides accounts for its ability, which is not shared with GPX, to protect membrane from lipid peroxidation, in cooperation with vitamin E. PHGPX was identified in a wide range of mammalian tissues-red blood cells being a notable exceptionand has been recently detected in human tumor-derived cells lines-MCF 7, K 562, HL 60, their doxorubicin resistant variant ADR R, and Hep G2after growth in selenium-sufficient medium. PHGPX specific activity in these cells ranged between 7-22 nmol/min/mg protein. The location of the enzyme was found to be mainly cytosolic, but 3-10% of the total activity was detected in the membrane fraction, whereas 15% of the total activity was detected in the nuclear fraction. The cytosolic enzyme was identified among the radiolabeled selenioproteins present in HEP G2 and

Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.

The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.

Correction of the N-Terminal Sequences of the Human Plastin Isoforms by Using Anchored Polymerase Chain Reaction: Identification of a Potential Calcium-Binding Domain

Plastins are a family of at least three cytoplasmic protein isoforms that are expressed differentially between cells of the hematopoietic lineages and cells of solid tissues. Expression of the L-plastin isoform appears to be restricted to replicating blood cells, and the two T-plastin isoforms appear to be restricted to replicating cells of solid tissues. However, L-plastin is induced in many human solid tumor-derived cells. We used the anchored polymerase chain reaction technique to amplify and clone the missing 5' ends of plastin mRNAs. We found that both plastin isoforms contain a potential calcium binding site near the N terminus.

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Plastins are a family of at least three cytoplasmic protein isoforms that are expressed differentially between cells of the hematopoietic lineages and cells of solid tissues. Expression of the L-plastin isoform appears to be restricted to replicating blood cells, and the two T-plastin isoforms appear to be restricted to replicating cells of solid tissues. However, L-plastin is induced in many human solid tumor-derived cells. We used the anchored polymerase chain reaction technique to amplify and clone the missing 5' ends of plastin mRNAs. We found that both plastin isoforms contain a potential calcium binding site near the N terminus.

Platelet Derived Growth Factor (PDGF) autocrine components in human tumor cell lines

Tumor cells may stimulate their own proliferation through an autocrine mechanism by simultaneously producing growth factors and growth factor receptors. We now report that numerous human tumor-derived cell lines simultaneously express the genes for platelet-derived growth factor (PDGF) A and B chains and the PDGF receptor (PDGF-R). Measurement of mRNA transcribed from these genes showed that among 16 malignant glioma cell lines tested, 15 expressed the PDGF A gene, 12 expressed the PDGF B gene, and 13 expressed the PDGF-R gene. Of three osteosarcoma lines, three expressed PDGF A, two expressed PDGF B, and three expressed PDGF-R. For eight malignant melanoma lines, seven expressed PDGF A, five expressed PDGF B, and three expressed PDGF-R genes. Thus, 13 of 16 malignant glioma, 3 of 3 osteosarcomas, and 3 of 8 malignant melanoma cell lines expressed the PDGF receptor gene and either or both PDGF genes. Five cell lines were tested for production of biologically active PDGF and PDGF receptor protein. Media conditioned by each of the five cell lines induced tyrosine phosphorylation of a protein identical in size to the PDGF receptor. These five cell lines also produced PDGF receptor protein as measured by Western blot analysis or metabolic labeling and immunoprecipitation using PDGF-R antibodies. The PDGF receptors of these cell lines were activated by human platelet PDGF or by recombinant AA or BB homodimers. Intracellular interaction of these receptors with the growth factor simultaneously produced may provide continuous stimulation to the proliferation of these cells.

Human gamma enolase: isolation of a cDNA clone and expression in normal and tumor tissues of human origin.

We have isolated and sequenced a cDNA clone encoding the human gamma enolase. Comparison of our cDNA sequence and the rat gamma enolase sequence revealed 97% homology at the level of amino acid sequence. The two coding regions were 91% homologous on the nucleotide level, whereas the 3' noncoding regions were much less homologous (32%). Further comparison of our cDNA sequence with the human alpha enolase revealed an 82% homology at the amino acid level and a 75% homology at the nucleotide level for the two coding regions, whereas the 3' nontranslated regions were only 30% homologous. Using a portion of the 3' nontranslated region of our cDNA, shown to be specific for human gamma enolase, a single 2.5 kb mRNA was detected in human brain tissue. This same gamma enolase message was also found in a number of human normal nonneuronal tissues, and in several human tumor-derived cell lines. Expression of the mRNA for the gamma enolase subunit should thus be used with caution when identifying the cells of neuronal or neuroendocrine origin.

Synergistic effect of 5-azacytidine and .GAMMA.-interferon or dimethyl sulfoxide on expression of HLA class I antigens by PLC-PRF-5 cells.

In order to elucidate the mechanism by which HLA antigens expression is induced or enhanced on the injured or transformed hepatocytes, we have made in vitro studies using human hepatic tumor-derived cell lines as a model system. In the present study, PLC-PRF-5 cells that have the integrated form of hepatitis B virus genome in DNA were treated with 5-azacytidine (5-azaC) in combination with gamma-interferon (IFN-gamma) or dimethyl sulfoxide (DMSO). HLA antigens on the cell surface were quantitated by using a modified cell-ELISA method. As a result, it was demonstrated that DMSO- or IFN-gamma-treatment enhanced expression of HLA class I antigens on the cell surface. In addition, enhanced expression of the antigens on PLC-PRF-5 cells treated with 5-azaC in combination with IFN-gamma or DMSO represented a synergistic effect of these inducers on HLA class I antigens expression although no changes in HLA antigens expression were induced after 5-azaC-treatment alone in short-term experiments. Furthermore, an indirect immunofluorescent analysis of hepatitis B surface antigen on the cells demonstrated increased expression of the antigen after 5-azaC-treatment alone. HLA class II antigens and hepatitis B core antigen were not induced even after those treatments and also not after a long-term experiment. These results might indicate possible modulation of HLA class I and hepatitis B virus antigens expression on the cultured cells by a DNA hypomethylating agent, 5-azaC.

Preneoplastic and neoplastic growth of xenotransplanted lung-derived human cell lines using deepithelialized rat tracheas

The human lung tumor-derived cell lines A549, Calu-1, Calu-3, HuT292, and SW900 and the transformed human bronchial epithelial cell line TBE-1, that was transfected with the v-Harvey-ras oncogene, were inoculated into deepithelialized Fisher 344 rat tracheas (5 X 10(5) cells/trachea). After the ends of the tracheas were sealed, the tracheas were transplanted into s.c. tissues of nude mice. In a parallel experiment, 1 X 10(6) cells from each of these cell lines were injected s.c. Histological examination of the tracheal transplants 2, 4, 8, 12, and 16 weeks after cell inoculation proved to be of greater usefulness than either clinical or histological observation of the s.c. injection sites. A549, Calu-1, and TBE-1 produced intratracheal neoplastic nodules as early as 2 weeks after cell inoculation. Calu-3, HuT292, and SW900 grew relatively slowly in the tracheas, and simple or stratified epithelia with slight or moderate atypia (preneoplastic lesions) were seen at 2 weeks. After the 4th week, they produced tumor nodules in the tracheal transplants, whereas no tumor cells could be seen at the s.c. injection sites. The human derivation of the cells was confirmed by in situ hybridization using human-specific DNA probes. The intratracheal inoculation and xenotransplantation of human-derived cell lines offers a time-saving alternative to the s.c. inoculation assay for tumorigenicity and is at the same time a potentially valuable approach to studying preneoplastic and neoplastic progression with human cell subpopulations.

Activation of pp60c-src protein kinase activity in human colon carcinoma.

The tyrosine-specific protein kinase activity of pp60c-src molecules obtained from human colon carcinoma tissues and tumor-derived cell lines was found to be elevated over that from normal colon tissues or cultures of normal colon mucosal cells. The elevated pp60c-src protein kinase activity in tumor tissues and in cultured colon carcinoma cells does not appear to result solely from an increase in the abundance of the c-src-encoded protein, suggesting that the specific activity of the pp60c-src tyrosine phosphotransferase is enhanced. These results raise the possibility that activation of the pp60c-src protein kinase may contribute to the genesis of human colon tumors.

Quisqualate selectively inhibits a brain peptisade which cleaves N-acetyl-L-aspartyl-L-glutamate in vitro

The expression of glutamate carboxypeptidase II (GCP II) is reduced in selective brain regions in schizophrenic patients. To investigate transcriptional mechanisms regulating the human GCP II gene, a 3460 bp DNA fragment comprised of the proximal 3228 bp of 5′ untranscribed sequence and first 232 bp of 5′ UTR portion of this gene was cloned into the mammalian luciferase reporter gene vector pGL3-Basic. Transfection assays in human astrocyte-derived SVG and human prostate tumor-derived LNCaP cells demonstrated that constructs with 3460, 1590 and 761 bp portions of 5′ region of human GCP II gene were able to drive the luciferase reporter gene. Additional deletion constructs showed that in the SVG cell line, constructs with 511 and 411 bp of GCP II gene fragments yielded highest transcriptional activity, with declining activity upon further removal of 5′ sequences. 15 bp of the promoter 5′ to a 225 bp GCP II fragment were essential for luciferase expression. Thus, in the SVG cells, the proximal 240 bp of the human GCP II promoter (232 bp of the 5′ UTR and 8 bp of 5′ untranscribed sequences) may represent the core promoter. Further, while a LyF-1 site lies within and overlaps a transcription start site in the 15 bp sequence, site-directed mutagenesis shows that LyF-1 is not the transcription initiator for the “TATA and CAAT” box lacking GCP II gene in the SVG cells. Finally, pattern differences in GCP II gene promoter expression in SVG and LNCaP cells suggest that sequences beyond 240 bp may be important for tissue-specific GCP II expression.

Inherent resistance of HeLa cell derivatives to paromomycin.

The human tumor-derived cell line HeLa S3 and nuclear and mitochondrial gene mutants derived from it are resistant to the aminoglycoside antibiotic, paromomycin (PAR). Other carcinoma-derived cells, SV40-transformed cells, and four human diploid fibroblast cell lines are all sensitive to PAR. Sensitivity is dependent on cell density, and at cell numbers greater than 400/cm2 sensitive cells will proliferate in PAR. The resistance to PAR is inherited in a dominant manner in cell-to-cell fusion hybrids, but is not transferred in cytoplast-to-cell fusions. PAR resistance is therefore encoded by a nuclear gene(s). Resistance to PAR is not caused by changes in the response of mitochondrial or cytoplasmic protein synthesis to PAR in vitro. The uptake of PAR is similar in resistant and sensitive cells, and dimethyl sulfoxide does not render resistant cells more sensitive. Thus, HeLa cell PAR resistance is unlike previously reported ribosomal mutations and may derive from differences in the intracellular metabolism of PAR.

Clonal variation of cadmium response in human tumor cell lines.

Subpopulations of human tumor-derived cell lines A101D, A204, and A549 were screened for Cd2+ cytotoxic response. Three of six A549, two of seven A101D, and four of seven A204 subpopulations were found to differ significantly from the parental line. A variant subpopulation of A101D (T3) was shown by flow cytometry to be comprised of cells having two distinct DNA histograms. One histogram, type 1, resembles that of normal human fibroblasts. The other, type 2, represents cells with one-third more DNA. Early passage T3 clonal populations were comprised primarily of type 1 cells. With passage, type 1 cells decreased relative to type 2 so that by passage 47 the culture was predominantly type 2. Correspondingly, the A101D T3 subpopulation became more Cd2+ sensitive with time in culture. Subclones having only type 1 DNA histograms were found to be Cd2+ resistant relative to subclones with type 2 histograms, and treatment of A101D T3 cultures having approximately equal amounts of type 1 and 2 cells with 2 microM Cd2+ resulted in the selection of type 1 cells. The enhanced Cd2+ resistance phenotype shown by A101D T3 type 1 cells correlated with reduced Cd2+ uptake and is not attributable to enhanced metallothionein synthesis.