Survey of interferon production and sensitivity in human cell lines

[This corrects the article on p. 102 in vol. 22.].     

Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines.

A number of mutant Chinese hamster ovary (CHO) cell lines resistant to the cytotoxic action of alpha-amanitin have been isolated. The alpha-amanitin sensitivity of the different mutant cell lines varied widely, but correlated well with the alpha-amanitin sensitivity of the RNA polymerase II activity in each of these mutant cell lines. In comparison with the RNA polymerase II of wild-type cells, three mutants, Ama39, Ama6, and Amal, required respectively 2- to 3-fold, 8- to 10-fold, and about 800-fold higher concentrations of alpha-amanitin for inhibition of their polymerase II activity. Determination of the equilibrium dissociation constants (KD) for complexes between 0-[3H]methyl-demethyl-gamma-amanitin and RNA polymearse II indicated that differences in alpha-amanitin sensitivity were reflected in differences in the ability of the enzymes to bind amanitin. Hybrids formed by fusion of mutants with cells of wild-type sensitivity contained both mutant and wild-type polymerase II activities. Thus, each of the different alpha-amanitin resistance mutations was expressed co-dominantly. A test for complementation between two of these mutations by measurement of both the alpha-amanitin sensitivity and the [3H]amanitin binding by RNA polymerase II in Ama6 X Amal hybrid cells did not reveal any wild-type RNA polymerase II activity. These data provide evidence that the mutation to alpha-amanitin resistance involves structural changes in the gene coding for the alpha-amanitin binding subunit of RNA polymerase II. These changes appear to account for the alpha-amanitin-resistant phenotypes of these mutant cells.     

Use of host cell reactivation of cisplatin-treated adenovirus 5 in human cell lines to detect repair of drug-treated DNA

This study demonstrates that whilst some DNA-repair deficiencies can be detected using host cell reactivation of cisplatin (CDDP)-treated adenovirus (Ad5), not all repair deficiencies affected replication of CDDP-treated Ad5 in human cells. A line of fibroblasts (XP25), derived from a patient with a UV-hypersensitive syndrome xeroderma pigmentosum (XP), was found, as previously reported [1], to be deficient in reactivating the treated virus when compared to the apparently repair-proficient human tumor cell lines established from bladder and ovarian carcinomas. However, a testicular teratoma cell line (SuSa), shown previously to be deficient in the repair of guanine-guanine (G-G) intrastrand crosslinks, adenine-guanine (A-G) intrastrand crosslinks and interstrand crosslinks [2], was found to reactivate the treated virus to a similar extent as the repair-proficient ovarian tumor cell line and the similarly repair-proficient RT112 cell line derived from a bladder carcinoma. Therefore, not all repair-deficient cell lines were deficient at CDDP-treated Ad5 reactivation. However, the HCR technique may still prove to be useful as a rapid screen for DNA-repair deficiencies in CDDP-sensitive cells of unknown repair capacity. A CDDP-sensitive ovarian tumor cell line (TR175) was deficient in reactivating CDDP-treated Ad5, whilst another ovarian cell line (TR170) of intermediate CDDP sensitivity reactivated the virus to a marginally higher extent than the other more CDDP-resistant repair proficient ovarian cell line (SKOV3). In addition, sublines of either the SuSa cells or the RT112 cells expressing approximately two-fold levels of resistance or increased sensitivity to CDDP, showed no change in their abilities to reactivate this CDDP-treated virus, compared to their parental lines. CDDP-treated Ad5 was also used as a lethal probe to obtain cell lines specifically deficient in DNA repair. One such deficient line (SKOV3-C3A), derived from the SKOV3 ovarian carcinoma cell line, displayed an unusual biphasic curve for reactivation of the CDDP-treated virus. Further cell lines derived in this novel manner may prove useful in analysing the genetics of CDDP-repair.     

A mutant of Listeria monocytogenes LO28 unable to induce an acid tolerance response displays diminished virulence in a murine model.

Exposing Listeria monocytogenes LO28 to sublethal pH induces protection against normally lethal pH conditions, a phenomenon known as the acid tolerance response. We identified a mutant, L. monocytogenes ATR1, which is incapable of inducing such tolerance, either against low pH or against any other stress tested. The virulence of this mutant was considerably decreased, suggesting that the acid tolerance response contributes to in vivo survival of L. monocytogenes.     

Hepatocyte growth factor has potent anti-proliferative activity in various tumor cell lines.

Hepatocyte growth factor (HGF) has potent mitogenic activity for mature hepatocytes and various normal epithelial cells. We now have evidence that HGF at 1-10 ng/ml, strongly inhibits the growth of HepG2 hepatocellular carcinoma cells, B6/F1 melanoma cells and KB squamous carcinoma cells. These tumor cells express high affinity receptors for HGF with a Kd of 25-28 pM, similar to findings with hepatocytes. HGF at 1-100 ng/ml had no significant cytolytic effect on tumor cells. Therefore, the anti-proliferative effect of HGF on tumor cells seems to be cytostatic, not cytolytic. As HGF apparently has bidirectional effects on cell growth, the possibility that it can serve as an anti-tumor agent merits attention.     

Two-dimensional analysis of proteins associated with heterogenous nuclear RNA in various animal cell lines.

The protein complement of heterogenous nuclear RNA . protein particles from human HeLA, mouse L and Chinese hamster (CHO) cells has been analysed by two-dimensional gel electrophoresis using the two techniques described by O'Farrell [J. Biol. Chem. (1975) 250, 4007--4021 and Cell (1977) 12, 1133--1142]. Over a hundred individual spots habe been reproducibly detected both L-[35S]methionine. Large similarities, especially in the 25 000--40 000 Mr cluster of basic protein, were found among these three mammalian species. As far as phosphoproteins are concerned, it was observed that the bands already described by one-dimensional gels [Eur. J. Biochem. (1978) 86, 301--310] with Mr values of 28 000, 30 000, 37 000 and 52 000 are resolved into about 15 individual spots, suggesting a corresponding number of distinct states of phosphorylation. It was also clearly demonstrated that phosphoproteins are unrelated to the major basic protein species. Particles of different size classes were analysed with respect to their content of individual proteins, both non-phosphorylated and phosphorylated. The most salient feature observed was that phosphoproteins become progressively more abundant with particles of increasing size. This raises the possibility that at least some of these phosphoproteins might belong to a nuclear structure to which hnRNA is normally bound.     

Metabolic enzyme diversity in different human thyroid cell lines and their sensitivity to gravitational forces

Many cancer cells show unique protein expression patterns. We used proteome technology including MS, free flow isoelectric focusing and Western blotting to determine current concentrations of metabolic enzymes in healthy and malignant human thyroid cells. Three different types of human thyroid cells were investigated after they had been cultured under equal conditions. MS revealed high quantities of glycolytic enzymes and moderate quantities of citric acid cycle enzymes in malignant FTC-133 cells with abnormal LDH B-chains, high quantities of glycolytic enzymes and marginal quantities of citric acid cycle enzymes in normal HTU-5 cells, and low quantities of glycolytic enzymes and marginal quantities of citrate cycle enzymes in malignant CGTH-W1 cells with abnormal LDH A-chains. When an alteration of gene expression activity was challenged physically by removing gravity forces, the concentrations of various glycolytic enzymes were changed in normal and malignant thyroid cells. However, the changes varied among the different cell types. Different cellular alignment of the enzymes could be one reason for the cell type-specific behavior as demonstrated by histological analysis of alpha-enolase.     

A mutant herpes simplex virus type 1 unable to express glycoprotein L cannot enter cells, and its particles lack glycoprotein H.

Herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) is essential for virus entry into cells and forms a hetero-oligomer with a newly described viral glycoprotein, gL. Normal folding, posttranslational processing, and intracellular transport of both gH and gL depend upon the coexpression of gH and gL in cells infected with vaccinia virus vectors (L. Hutchinson, H. Browne, V. Wargent, N. Davis-Poynter, S. Primorac, K. Goldsmith, A. C. Minson, and D. C. Johnson, J. Virol. 66:2240-2250, 1992). Homologs of gH and gL have been found in herpesviruses of all subgroups, and thus it appears likely that the gH-gL complex serves a highly conserved function during herpesvirus penetration into cells. To examine the role of gL in the infectious cycle of HSV-1, a mutant HSV-1 unable to express gL was constructed by inserting a lacZ gene cassette into the coding sequences of the UL1 (gL) gene. Because gL was found to be essential for virus replication, cell lines capable of expressing gL were constructed to complement the virus mutant. In the absence of gL, virus particles were produced, and these particles reached the cell surface; however, gL-negative particles purified from infected cells were also deficient in gH. Mutant virions lacking gH and gL were able to adsorb onto cells but were unable to enter cells and initiate an infection. Further, the role of gL in fusion of infected cells was reexamined. A mutation in HSV-1 (804) which produces the syncytial phenotype had previously been mapped to a region of the HSV-1 genome which includes the UL1 gene and no other open reading frame. However, in contrast to this previous report, we found that the syncytial mutation in 804 affects the UL53 gene, which encodes gK, a gene commonly mutated in syncytial viruses.     

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.     

Temperature-dependent growth rates and gene expression patterns of various medaka Oryzias latipes cell lines derived from different populations

Medaka Oryzias latipes has several geographically and genetically distinct populations. We examined temperature acclimation response in various medaka cell lines derived from different populations. Measurement of cell growth at various temperatures suggested that 15°C was the permissive growth temperature in all cell lines from the Northern Japanese and East Korean populations, but not in those from the Southern Japanese population and medaka-related species Oryzias celebensis, which inhabits a tropical zone. RT-PCR for 102 temperature-responsive genes, previously reported in other species, revealed that the accumulated mRNA level of a gene encoding HSP47 was lower at 25°C than at 33°C, and vice versa for 12 genes including IκBα and Rab-1c, in OLHNI-1 cell line from the Northern Japanese population. Further analysis by real-time PCR demonstrated that the accumulated mRNA levels of IκBα and Rab-1c in OLHNI-1 and OLSOK-e7 cell lines from the East Korean population were increased when the culture temperature was shifted from 33 to 15°C, but not in OLHdrR-e3 cell line from the Southern Japanese population. Since IκBα and Rab-1c are related to the NFκB cascade and endoplasmic reticulum-to-Golgi transport, respectively, it is inferred that immune responses and intracellular transport are possibly critical to temperature adaptation for medaka. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.     

Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter- and intramolecular recombination events.     

A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.     

delta-Aminolevulinic acid synthesis in a Cyanidium caldarium mutant unable to make chlorophyll a and phycobiliproteins.

Wild-type cells of the unicellular rhodophyte, Cyanidium caldarium, synthesize chlorophyll a, phycobiliproteins, and heme from δ-aminolevulinic acid during light-dependent chloroplast development but are unable to make photosynthetic pigments in the dark. C. caldarium, mutant GGB-Y, is an obligate heterotroph which, in the light, produces a chloroplast devoid of photosynthetic pigments. The present investigation has shown that δ-aminolevulinic acid is synthesized in cells of mutant GGB-Y incubated with levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase (the second enzyme in the porphyrin biosynthetic pathway). In vivo, cells of mutant GGB-Y preferentially incorporated C₁ of glutamate and α-ketoglutarate into the C₅ fragment (formaldehyde) of δ-aminolevulinic acid after alkaline periodate degradation. This suggested that δ-aminolevulinic acid arises directly from the carbon skeleton of glutamate and α-ketoglutaric acid. The pattern of incorporation of C₃, C₄, and C₅ of α-ketoglutarate into the C₁–C₄ (succinic acid) fragment of δ-aminolevulinic acid after alkaline periodate degradation was consistent with the origin of δ-aminolevulinic acid from a five-carbon precursor. C₁ and C₂ of glycine and C₂ and C₃ of succinate were incorporated into both the formaldehyde and succinate fragments of δ-aminolevulinic acid in a manner inconsistent with condensation of glycine and succinyl CoA by δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the porphyrin pathway in animals and bacteria. Extracts of the soluble protein from cells of mutant GGB-Y displayed a Soret band at 410 nm indicating the presence of hemoproteins. This shows that mutant GGB-Y cells synthesize heme. The respiration of radiolabeled glutamate, α-ketoglutarate, and glycine to ¹⁴CO₂ is consistent with the existence of mitochondrial cytochromes in cells of mutant GGB-Y and with the ability of the mutant to synthesize δ-aminolevulinic acid. The present results suggest that δ-aminolevulinic acid is synthesized directly from glutamate or α-ketoglutarate and that this is the only process by which the rate-limiting intermediate in the porphyrin pathway is synthesized in C. caldarium. If correct, the rate-limiting, regulative enzyme in the biosynthetic pathway for synthesis of chlorophyll a, bile pigment (phycocyanobilin), and heme must have been completely different in the evolutionary antecedents of modern-day plants and animals.     

Characterization of a fission yeast mutant which displays defects in cell wall integrity and cytokinesis.

The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC). The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls. The arrested cells display a disoriented alignment of cytoplasmic microtubules. When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick. Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix. rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S. pombe, in the pathway of cell wall integrity and cytokinesis.     

Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins.

Adenovirus mutants containing genetic alterations in the gene encoding the E1B 19,000-molecular-weight (19K) tumor antigen induce the degradation of host cell chromosomal DNA (deg phenotype) and enhanced cytopathic effect (cyt phenotype) after infection of HeLa and KB cells. The deg and cyt phenotypes are a consequence of viral early gene expression in the absence of the E1B 19K protein. The role of the E1A proteins in induction of the cyt and deg phenotypes was investigated by constructing E1A-E1B double mutant viruses. Viruses were constructed to express the individual E1A 13S, 12S, or 9S cDNA genes in the presence of a mutation in the gene encoding the E1B 19K tumor antigen. Expression of either the 13S or 12S E1A proteins in the absence of functional E1B 19K protein produced the deg and cyt phenotypes. In contrast, a virus which expressed exclusively the 9S E1A gene product in the absence of the E1B 19K gene product did not induce the deg and cyt phenotypes, even at high multiplicities of infection. Therefore, both the 13S and 12S E1A gene products could directly or indirectly cause the deg and cyt phenotypes during infection of HeLa cells with an E1B 19K gene mutant virus. Furthermore, the deg phenotype was found to be host cell type specific, occurring in HeLa and KB cells but not in growth-arrested human WI38 cells. These results indicate that expression of the E1A trans-activating and transforming proteins is necessary for the induction of the cyt and deg phenotypes and that host cell factors also play a role.