CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production

NY-ESO-1 is a tumor-specific shared antigen with distinctive immunogenicity. Both CD8+ T cells and class-switched Ab responses have been detected from patients with cancer. In this study, a CD4+ T cell line was generated from peripheral blood mononuclear cells of a melanoma patient and was shown to recognize NY-ESO-1 peptides presented by HLA-DP4, a dominant MHC class II allele expressed in 43–70% of Caucasians. The ESO p157–170 peptide containing the core region of DP4-restricted T cell epitope was present in a number of tumor cell lines tested and found to be recognized by both CD4+ T cells as well as HLA-A2-restricted CD8+ T cells. Thus, the ESO p157–170 epitope represents a potential candidate for cancer vaccines aimed at generating both CD4+ and CD8+ T cell responses. More importantly, 16 of 17 melanoma patients who developed Ab against NY-ESO-1 were found to be HLA-DP4-positive. CD4+ T cells specific for the NY-ESO-1 epitopes were generated from 5 of 6 melanoma patients with NY-ESO-1 Ab. In contrast, no specific DP4-restricted T cells were generated from two patients without detectable NY-ESO-1 Ab. These results suggested that NY-ESO-1-specific DP4-restricted CD4+ T cells were closely associated with NY-ESO-1 Ab observed in melanoma patients and might play an important role in providing help for activating B cells for NY-ESO-1-specific Ab production.

Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α TubulinV⃞

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.

Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation

Human telomerase, a cellular reverse transcriptase (hTERT), is a nuclear ribonucleoprotein enzyme complex that catalyzes the synthesis and extension of telomeric DNA. This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells, suggesting that telomerase plays an important role in cellular immortalization and tumorigenesis. Terminal maturation of tumor cells has been associated with the repression of telomerase activity. Using maturation-sensitive and -resistant NB4 cell lines, we analyzed the pattern of telomerase expression during the therapeutic treatment of acute promyelocytic leukemia (APL) by retinoids. Two pathways leading to the down-regulation of hTERT and telomerase activity were identified. The first pathway results in a rapid down-regulation of telomerase that is associated with retinoic acid receptor (RAR)-dependent maturation of NB4 cells. Furthermore, during NB4 cell maturation, obtained independently of RAR by retinoic X receptor (RXR)-specific agonists (rexinoids), no change in telomerase activity was observed, suggesting that hTERT regulation requires a specific signaling and occurs autonomously. A second pathway of hTERT regulation, identified in the RAR-responsive, maturation-resistant NB4-R1 cell line, results in a down-regulation of telomerase that develops slowly during two weeks of all-trans retinoic acid (ATRA) treatment. This pathway leads to telomere shortening, growth arrest, and cell death, all events that are overcome by ectopic expression of hTERT. These findings demonstrate a clear and full dissociation between the process of tumor cell maturation and the regulation of hTERT mRNA expression and telomerase activity by retinoids. We propose telomerase expression as an efficient and selective target of retinoids in the therapy of tumors.

A low threshold level of expression of mutant-template telomerase RNA inhibits human tumor cell proliferation

The ribonucleoprotein telomerase synthesizes telomeric DNA by copying an intrinsic RNA template. In most cancer cells, telomerase is highly activated. Here we report a telomerase-based antitumor strategy: expression of mutant-template telomerase RNAs in human cancer cells. We expressed mutant-template human telomerase RNAs in prostate (LNCaP) and breast (MCF-7) cancer cell lines. Even a low threshold level of expression of telomerase RNA gene constructs containing various mutant templates, but not the control wild-type template, decreased cellular viability and increased apoptosis. This occurred despite the retention of normal levels of the endogenous wild-type telomerase RNA and endogenous wild-type telomerase activity and unaltered stable telomere lengths. In vivo tumor xenografts of a breast cancer cell line expressing a mutant-template telomerase RNA also had decreased growth rates. Therefore, mutant-template telomerase RNAs exert a strongly dominant-negative effect on cell proliferation and tumor growth. These results support the potential use of mutant-template telomerase RNA expression as an antineoplastic strategy.

Short tandem repeat profiling provides an international reference standard for human cell lines

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.

Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.

Isolation and characterization of mutant cell lines defective in transforming growth factor beta signaling.

To isolate and characterize effector molecules of the transforming growth factor beta (TGFbeta) signaling pathway we have used a genetic approach involving the generation of stable recessive mutants, defective in their TGFbeta signaling, which can subsequently be functionally complemented to clone the affected genes. We have generated a cell line derived from a hypoxanthine-guanine phosphoribosyltransferase negative (HPRT-) HT1080 clone that contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to a TGFbeta-responsive promoter. This cell line proliferates or dies in the appropriate selection medium in response to TGFbeta. We have isolated three distinct TGFbeta-unresponsive mutants following chemical mutagenesis. Somatic cell hybrids between pairs of individual TGFbeta-unresponsive clones reveal that each is in a distinct complementation group. Each mutant clone retains all three TGFbeta receptors yet fails to induce a TGFbeta-inducible luciferase reporter construct or TGFbeta-mediated plasminogen activator inhibitor-1 (PAI-1) expression. Two of the three have an attenuated TGFbeta-induced fibronectin response, whereas in the other mutant the fibronectin response is intact. These TGFbeta-unresponsive cells should allow selection and identification of signaling molecules through functional complementation.

Infectivity of chimeric human T-cell leukemia virus type I molecular clones assessed by naked DNA inoculation.

Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.

RIG-E, a human homolog of the murine Ly-6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell.

In vivo all-trans-retinoic acid (ATRA), a differentiation inducer, is capable of causing clinical remission in about 90% of patients with acute promyelocytic leukemia (APL). The molecular basis for the differentiation of APL cells after treatment with ATRA remains obscure and may involve genes other than the known retinoid nuclear transcription factors. We report here the ATRA-induced gene expression in a cell line (NB4) derived from a patient with APL. By differential display-PCR, we isolated and characterized a novel gene (RIG-E) whose expression is up-regulated by ATRA. The gene is 4.0 kb long, consisting of four exons and three introns, and is localized on human chromosome region 8q24. The deduced amino acid sequence predicts a cell surface protein containing 20 amino acids at the N-terminal end corresponding to a signal peptide and an extracellular sequence containing 111 amino acids. The RIG-E coded protein shares some homology with CD59 and with a number of growth factor receptors. It shares high sequence homology with the murine LY-6 multigene family, whose members are small cysteine-rich proteins differentially expressed in several hematopoietic cell lines and appear to function in signal transduction. It seems that so far RIG-E is the closest human homolog of the LY-6 family. Expression of RIG-E is not restricted to myeloid differentiation, because it is also present in thymocytes and in a number of other tissues at different levels.

p53 as a target for cancer vaccines: recombinant canarypox virus vectors expressing p53 protect mice against lethal tumor cell challenge.

The p53 protein is an attractive target for immunotherapy, because mutations in the p53 gene are the most common genetic alterations found in human tumors. These mutations result in high levels of p53 protein in the tumor cell, whereas the expression level of wild-type p53 in nonmalignant tissue is usually much lower. Several canarypox virus recombinants expressing human or murine p53 in wild-type or mutant form were constructed. Immunization with these viruses protected BALB/c mice from a challenge with an isogenic and highly tumorigenic mouse fibroblast tumor cell line expressing high levels of mutant p53. The tumor protection was equally effective regardless of whether wild-type or mutant p53 was used for the immunization, indicating that the immunologic response was not dependent on any particular p53 mutation and that immunization with this live virus vaccine works effectively against mutant p53 protein expressed in a tumor cell. In tumors escaping immunologic rejection, the expression of the p53 protein was commonly down-regulated.

Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors.

Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases.

Correlation between the effects of bombesin antagonists on cell proliferation and intracellular calcium concentration in Swiss 3T3 and HT-29 cell lines.

Bombesin (BN) acts as an autocrine mitogen in various human cancers. Several pseudononapeptide BN-(6-14) analogs with a reduced peptide bond between positions 13 and 14 have been shown to suppress the mitogenic activity of BN or gastrin-releasing peptide (GRP) when assessed by radioreceptor or proliferation assays and may have significant clinical applications. The search for potent and safe BN antagonists requires the evaluation of a large series of analogs in radioreceptor and proliferation assays. In this paper, we report that the ability of BN analogs to inhibit BN-induced calcium transients in Swiss 3T3 cells shows a high correlation with their inhibitory potency as evaluated by classical proliferation tests. The assay of calcium transients allows a rapid characterization of new BN analogs (in terms of minutes rather than days) and can be adapted as a labor and cost-effective screening step in the selection of potentially relevant BN antagonists for further characterization in cell proliferation systems. We also observed that results from the assay of calcium transients in Swiss 3T3 cells can be correlated with the results of the proliferative response in HT-29 cells, a cell line that does not seem to use the same early transmembrane ionic signal system. This result suggests that the calcium pathway is not mandatory for triggering cell division by the BN receptor.

Characterization of GMP-17, a granule membrane protein that moves to the plasma membrane of natural killer cells following target cell recognition.

Cytotoxic lymphocytes are characterized by their inclusion of cytoplasmic granules that fuse with the plasma membrane following target cell recognition. We previously identified a cytotoxic granule membrane protein designated p15-TIA-1 that is immunochemically related to an RNA-recognition motif (RRM)-type RNA-binding protein designated p40-TIA-1. Although it was suggested that p15-TIA-1 might be derived from p40-T1A-1 by proteolysis, N-terminal amino acid sequencing of p15-TIA-1 immunoaffinity purified from a natural killer (NK) cell line by using monoclonal antibody (mAb) 2G9 revealed that p15-T1A-1 is identical to the deduced amino acid sequence of NKG7 and GIG-1, cDNAs isolated from NK cells and granulocyte-colony-stimulating factor-treated mononuclear cells, respectively. Epitope mapping revealed that mAb 2G9 recognizes the C terminus of p15-T1A-1 and p40-T1A-1. The deduced amino acid sequence of p15-T1A-1/NKG7/GIG-1 predicts that the protein possesses four transmembrane domains, and immuno-electron microscopy localizes the endogenous protein to the membranes of cytotoxic granules in NK cells. Given its subcellular localization, we propose to rename-this protein GMP-17, for granule membrane protein of 17 kDa. Immunofluorescence microscopy of freshly isolated NK cells confirms this granular localization. Target cell-induced NK cell degranulation results in translocation of GMP-17 from granules to the plasma membrane, suggesting a possible role for GMP-17 in regulating the effector function of lymphocytes and neutrophils.

Angiotensin II type 2 receptor mediates programmed cell death.

The function of the recently discovered angiotensin II type 2 (AT2) receptor remains elusive. This receptor is expressed abundantly in fetus, but scantily in adult tissues except brain, adrenal medulla, and atretic ovary. In this study, we demonstrated that this receptor mediates programmed cell death (apoptosis). We observed this effect in PC12W cells (rat pheochromocytoma cell line) and R3T3 cells (mouse fibroblast cell line), which express abundant AT2 receptor but not AT1 receptor. The cellular mechanism appears to involve the dephosphorylation of mitogen-activated protein kinase (MAP kinase). Vanadate, a protein-tyrosine-phosphatase inhibitor, attenuated the dephosphorylation of MAP kinases by the AT2 receptor and restored the apoptotic changes. Antisense oligonucleotide to MAP kinase phosphatase 1 inhibited the AT2 receptor-mediated MAP kinase dephosphorylation and blocked the AT2 receptor-mediated apoptosis. These results suggest that protein-tyrosine-phosphatase, including MAP kinase phosphatase 1 activated by the AT2 receptor, is involved in apoptosis. We hypothesize that this apoptotic function of the AT2 receptor may play an important role in developmental biology and pathophysiology.