Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins.

The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.

Human neoplasms elicit multiple specific immune responses in the autologous host.

Expression of cDNA libraries from human melanoma, renal cancer, astrocytoma, and Hodgkin disease in Escherichia coli and screening for clones reactive with high-titer IgG antibodies in autologous patient serum lead to the discovery of at least four antigens with a restricted expression pattern in each tumor. Besides antigens known to elicit T-cell responses, such as MAGE-1 and tyrosinase, numerous additional antigens that were overexpressed or specifically expressed in tumors of the same type were identified. Sequence analyses suggest that many of these molecules, besides being the target of a specific immune response, might be of relevance for tumor growth. Antibodies to a given antigen were usually confined to patients with the same tumor type. The unexpected frequency of human tumor antigens, which can be readily defined at the molecular level by the serological analysis of autologous tumor cDNA expression cloning, indicates that human neoplasms elicit multiple specific immune responses in the autologous host and provides diagnostic and therapeutic approaches to human cancer.

Human naive and memory T lymphocytes differ in telomeric length and replicative potential.

The present study has assessed the replicative history and the residual replicative potential of human naive and memory T cells. Telomeres are unique terminal chromosomal structures whose length has been shown to decrease with cell division in vitro and with increased age in vivo for human somatic cells. We therefore assessed telomere length as a measure of the in vivo replicative history of naive and memory human T cells. Telomeric terminal restriction fragments were found to be 1.4 +/- 0.1 kb longer in CD4+ naive T cells than in memory cells from the same donors, a relationship that remained constant over a wide range of donor age. These findings suggest that the differentiation of memory cells from naive precursors occurs with substantial clonal expansion and that the magnitude of this expansion is, on average, similar over a wide range of age. In addition, when replicative potential was assessed in vitro, it was found that the capacity of naive cells for cell division was 128-fold greater as measured in mean population doublings than the capacity of memory cells from the same individuals. Human CD4+ naive and memory cells thus differ in in vivo replicative history, as reflected in telomeric length, and in their residual replicative capacity.

Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo.

Recruitment of antigen-specific tumor-infiltrating lymphocytes (TILs) is a major goal for immunotherapy of malignant tumours. We now describe that T-cell-activating superantigens targeted to a tumor by monoclonal antibodies induced large numbers of pseudospecific TILs and eradication of micrometastases. As a model for tumor micrometastases, syngeneic B16 melanoma cells transfected with the human colon carcinoma antigen C215 were injected intravenously into C57BL/6 mice and therapy with an anti-C215 Fab fragment-staphylococcal enterotoxin A (C215Fab-SEA) fusion protein reacting with the C215 antigen was initiated when visible lung metastases were established. More than 90% reduction of the number of lung metastases was observed when mice carrying 5-day-old established lung metastases were treated with C215Fab-SEA. The antitumor effect of C215Fab-SEA was shown to be T-cell-dependent since no therapeutic effect was seen in T-cell-deficient nude mice. Depletion of T-cell subsets by injection of monoclonal antibody demonstrated that CD8+ cells were the most prominent effector cells although some contribution from CD4+ cells was also noted. C215Fab-SEA treatment induced massive tumor infiltration of CD4+ and CD8+ T cells, while only scattered T cells were observed in untreated tumors. SEA treatment alone induced a slight general inflammatory response in the lung parenchyme, but no specific accumulation of T cells was seen in the tumor. TILs induced by C215Fab-SEA were mainly CD8+ but a substantial number of CD4+ cells were also present. Immunohistochemical analysis showed strong production of the tumoricidal cytokines tumor necrosis factor alpha and interferon gamma in the tumor. Thus, the C215Fab-SEA fusion protein targets effector T lymphocytes to established tumors in vivo and provokes a strong local antitumor immune response.

Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection.

The beta 1-6 structure of N-linked oligosaccharides, formed by beta-1,6-N-acetylglucosaminyltransferase (GnT-V), is associated with metastatic potential. We established a highly metastatic subclone, B16-hm, from low metastatic B16-F1 murine melanoma cells. The gene encoding beta-1,4-N-acetylglucosaminyltransferase (GnT-III) was introduced into the B16-hm cells, and three clones that stably expressed high GnT-III activity were obtained. In these transfectants, the affinity to leukoagglutinating phytohemagglutinin was reduced, whereas the binding to erythroagglutinating phytohemagglutinin was increased, indicating that the level of beta 1-6 structure was decreased due to competition for substrate between intrinsic GnT-V and ectopically expressed GnT-III. Lung metastasis after intravenous injection of the transfectants into syngeneic and nude mice was significantly suppressed, suggesting that the decrease in beta 1-6 structure suppressed metastasis via a mechanism independent of the murine system. These transfectants also displayed decreased invasiveness into Matrigel and inhibited cell attachment to collagen and laminin. Cell growth was not affected. Our results demonstrate a causative role for beta 1-6 branches in invasion and cell attachment in the extravasation stage of metastasis.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.

Ação da proteína amiloide sérica A em melanomas

Concentrações séricas basais da proteína amiloide sérica A (SAA) estão significativamente aumentadas em pacientes com câncer e alguns autores sugerem uma relação causal. Trabalho anterior do grupo mostrou que a SAA induz a proliferação de duas linhagens de glioblastoma humano e afeta os processos de invasividade in vitro, sustentando um papel pró-tumoral para esta proteína. Com base nesse trabalho, investigamos a abrangência dos efeitos de SAA para outro tipo de célula tumoral e para isso escolhemos um painel de linhagens de melanoma humano e uma linhagem primária obtida a partir de aspirado de linfonodo de paciente com melanoma, por nós isolada. Observamos que apesar da célula precursora de melanomas, isto é, melanócito, não produzir SAA, todas as linhagens de melanoma produziram a proteína e expressaram alguns dos seus receptores. Além disso, quando estas células foram estimuladas com SAA houve uma inibição da proliferação em tempos curtos de exposição (48 horas) e efeitos citotóxicos após um tempo maior (7 dias). A SAA também afetou processos de invasividade e a produção das citocinas IL-6, IL-8 e TNF-α. Aos avaliarmos o efeito da SAA na interação das células de melanoma com células do sistema imune, vimos que a SAA ativou uma resposta imune anti-tumoral aumentando a expressão de moléculas co-estumolatórias, como CD69 e HLA-DR, e sua função citotóxica. Ainda, vimos que a produção de TNF-α, IFN-γ, IL-10, IL-1β e IL-8 estimuladas por SAA podem contribuir com os efeitos desta. De forma geral estes resultados nos levam a crer que a SAA tem atividade anti-tumoral em melanomas. Finalizando, com base na importância do desenvolvimento da resistência às terapias atuais para o melanoma, observamos que em células resistentes ao PLX4032, um inibidor de BRAF, os efeitos imunomodulatórios induzidos pela SAA estão abolidos, possivelmente identificando um novo componente da resistência.

A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.

Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: Null mutations in the PDHX gene associated with unusual expression of the testis-specific PDHA2 gene in her somatic cells

Human pyruvate dehydrogenase complex (PDC) catalyzes a key step in the generation of cellular energy and is composed by three catalytic elements (E1, E2, E3), one structural subunit (E3-binding protein), and specific regulatory elements, phosphatases and kinases (PDKs, PDPs). The E1α subunit exists as two isoforms encoded by different genes: PDHA1 located on Xp22.1 and expressed in somatic tissues, and the intronless PDHA2 located on chromosome 4 and only detected in human spermatocytes and spermatids. We report on a young adult female patient who has PDC deficiency associated with a compound heterozygosity in PDHX encoding the E3-binding protein. Additionally, in the patient and in all members of her immediate family, a full-length testis-specific PDHA2 mRNA and a 5′UTR-truncated PDHA1 mRNA were detected in circulating lymphocytes and cultured fibroblasts, being bothmRNAs translated into full-length PDHA2 and PDHA1 proteins, resulting in the co-existence of both PDHA isoforms in somatic cells.Moreover, we observed that DNA hypomethylation of a CpG island in the coding region of PDHA2 gene is associatedwith the somatic activation of this gene transcription in these individuals. This study represents the first natural model of the de-repression of the testis-specific PDHA2 gene in human somatic cells, and raises some questions related to the somatic activation of this gene as a potential therapeutic approach for most forms of PDC deficiency.

Antiproliferative and phenotype-transforming antitumor agents derived from cysteine

Selective destruction of malignant tumor cells without damaging normal cells is an important goal for cancer chemotherapy in the 21st century. Differentiating agents that transform cancer cells to either a nonproliferating or normal phenotype could potentially be tissue-specific and avoid side effects of current drugs. However, most compounds that are presently known to differentiate cancer cells are histone deacetylase inhibitors that are of low potency or suffer from low bioavailability, rapid metabolism, reversible differentiation, and nonselectivity for cancer cells over normal cells. Here we describe 36 nonpeptidic compounds derived from a simple cysteine scaffold, fused at the C-terminus to benzylamine, at the N-terminus to a small library of carboxylic acids, and at the S-terminus to 4-butanoyl hydroxamate. Six compounds were cytotoxic at nanomolar concentrations against a particularly aggressive human melanoma cell line (MM96L), four compounds showed selectivities of greater than or equal to5:1 for human melanoma over normal human cells (NFF), and four of the most potent compounds were further tested and found to be cytotoxic for six other human cancer cell lines (melanomas SK-MEL-28, DO4; prostate DU145; breast MCF-7; ovarian JAM, CI80-13S). The most active compounds typically caused hyperacetylation of histones, induced p21 expression, and reverted phenotype of surviving tumor cells to a normal morphology. Only one compound was given orally at 5 mg/kg to healthy rats to look for bioavailaiblity, and it showed reasonably high levels in plasma (C-max 6 mug/mL, T-max 15 min) for at least 4 h. Results are sufficiently promising to support further work on refining this and related classes of compounds to an orally active, more tumor-selective, antitumor drug.

Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro

Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast-melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype. (c) 2005 Elsevier Inc. All rights reserved.

Spontaneous and UV radiation-induced multiple metastatic melanomas in Cdk4(R24C/R24C)/TPras mice

Human melanoma susceptibility is often characterized by germ-line inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UV radiation (UVR) dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0% to 57%. Here, we report that activated Cdk4 cooperates with activated Hras to enhance susceptibility to melanoma in mice. Whereas UVR treatment failed to induce melanomas in Cdk4(R24C/R24C) mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4(R24C/R24C)/TPras animals compared with TPras alone. This increased penetrance was dependent on the threshold of Cdk4 activation as Cdk4(R24C/+)/TPras animals did not show an increase in UVR-induced melanoma penetrance compared with TPras alone. In addition, Cdk4(R24C/R24C)/TPras mice invariably developed multiple lesions, which occurred rarely in TPras mice. These results indicate that germ-line defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4(R24C/R24C)/TPras tumors were comparable histopathologically but the latter were larger and more aggressive and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4(R24C/R24C)/TPras mice, but not in TPras mice, readily metastasized to regional lymph nodes. Thus, it seems that in the mouse, Hras activation initiates UVR-induced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors.

Design, Synthesis, Potency and Cytoselectivity of Anticancer Agents Derived by Parallel Synthesis from alpha-Aminosuberic Acid

Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 mu M), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 mu M). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.