Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutinin-stimulated cells from elderly donors.

Using cDNA probes to human interleukin 2 (IL2) and interleukin 2 receptor (IL2R), the amount of IL2 and IL2R mRNA produced by PHA stimulated peripheral blood mononuclear cells from young (less than 40 yr) and old (greater than 60 yr) donors was quantitated. Stimulated cell cultures from each individual were also examined for proliferative ability, expression of membrane IL2R, membrane IL2R density, and for the amount of IL2R shed into the culture supernatant. Induction of IL2 and IL2R mRNAs were decreased in cells from elderly individuals, as were the levels of IL2 secretion, the percentage of IL2R+ T cells and the density of membrane IL2R per cell. The results suggest that decreased expression of both IL2 and IL2R mRNA contributes to the low synthesis of IL2 and membrane IL2R, respectively, and is partially responsible for the diminished proliferative activity observed in lymphocytes from the elderly.

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2.

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.

Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines.

Pharmacological inhibitors of the anabolic enzyme, fatty acid synthase (FAS), including the natural product cerulenin and the novel compound c75, are selectively cytotoxic to cancer cells via induction of apoptosis, apparently related to the tumor cell phenotype of abnormally elevated fatty acid synthetic metabolism. As part of a larger effort to understand the immediate downstream effect of FAS inhibition that leads to apoptosis, the effects of these inhibitors on cell cycle progression were examined. Both FAS inhibitors produce rapid, profound inhibition of DNA replication and S phase progression in human cancer cells. The dose responses for fatty acid synthesis inhibition and DNA synthesis inhibition are similar. The kinetics of both effects are rapid, with fatty acid synthesis inhibition occurring within 30 min and DNA synthesis inhibition occurring within 90 min of drug exposure. Meanwhile, apoptotic changes are not detected until 6 h or later after inhibitor exposure. Fatty acid synthetic pathway activity and the magnitude of DNA synthesis inhibition by FAS inhibitors are increased in parallel by withdrawal of lipid-containing serum from the cultures. The mechanism of DNA synthesis inhibition by cerulenin is indirect, because expression of certain viral oncogenes rescues DNA synthesis/S phase progression in cerulenin-exposed cells. The data suggest a direct linkage at a regulatory level, between fatty acid synthesis and DNA synthesis in proliferating tumor cells.

The alpha-glucosidase I inhibitor castanospermine alters endothelial cell glycosylation, prevents angiogenesis, and inhibits tumor growth.

The development of drugs that target the tumor neovasculature may hold promise in inhibiting tumor growth. Experiments in vivo with castanospermine, an inhibitor of the glucosidases that convert protein N-linked high mannose carbohydrates to complex oligosaccharides, resulted in significant inhibition of tumor growth in nude mice. Angiogenesis to basic fibroblast growth factor in castanospermine-treated C57/BL mice was similarly reduced. Endothelial cell proliferation, invasion of basement membrane, and differentiation are crucial steps during neovascularization. In vitro differentiation models using Matrigel and postconfluent cultures of endothelial cells were used to study the effects of glycosidase inhibitors on endothelial cell behavior. FACS analysis of cell surface oligosaccharides using either Concanavalin A or L-phytohemagglutinin lectins confirmed an increase in high mannose groups and a decrease in tri- and tetra antennary beta-linked galactose-N-acetylglucosamine on mannose residues of Asn-linked oligosaccharides upon drug treatment. Castanospermine and the glucosidase inhibitor N-methyldeoxynojirimycin prevented the morphological differentiation of endothelial cells in vitro. These compounds did not alter the proliferation of cultured endothelial cells or their ability to attach to various extracellular matrix molecules. However, the cells showed a reduced ability to migrate and to invade basement membrane gels in vitro and an increased tendency to form aggregates that was inhibitable by D-mannose. These studies suggest that certain cell surface oligosaccharides are required for angiogenesis and that glucosidase inhibitors that alter these structures on endothelial cells are able to inhibit tumor growth.

Progression of hormone-dependent adenocarcinoma cells to hormone-independent spindle carcinoma cells in vitro in a clonal spontaneous rat mammary tumor cell line.

A hormone-dependent, clonal carcinoma cell line, designated RM22-F5, was derived from a malignant mammary mixed tumor spontaneously arising in an outbred old female Wistar rat. These cells expressed keratin and desmosomal protein and formed epithelial monolayers in a growth factor and hormone-supplemented medium (LHC-8) containing 10% fetal bovine serum (E-type cells). Cells subcultured for 6 to 8 passages in RPMI 1640 medium containing 10% fetal bovine serum without supplements appeared to be fibroblastic and expressed vimentin (F-type cells). The shift to a fibroblast-like morphology was associated with a more malignant phenotype which included rapid, hormone-independent growth and invasive sarcoma-like character in nude mice. F-type cells were no longer able to express their original epithelial phenotype in LHC-8 medium. Cytogenetic analysis revealed that both E- and F-type cells had essentially the same karyotype. Analysis of PCR-amplified DNA further demonstrated a point mutation of the H-ras-1 gene at codon 12 and loss of the normal H-ras-1 allele in both cell types. Genetic tagging of E-type cells with the neomycin-resistance gene resulted in the generation of F-type cells with neomycin resistance in RPMI 1640 medium, suggesting that F-type cells are a malignant variant of E-type cells arising during in vitro culture. Somatic cell fusion between E- and F-type cells revealed that with most hybrid clones tested, the fibroblast-like phenotype was greatly suppressed. These results suggest that an irreversible phenotypic transition, representative of tumor progression from hormone-dependent adenocarcinoma to more malignant hormone-independent spindle carcinoma cells, is a recessive event and may involve loss of a suppressor function.

Malonyl-coenzyme-A is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts.

A biologically aggressive subset of human breast cancers and other malignancies is characterized by elevated fatty-acid synthase (FAS) enzyme expression, elevated fatty acid (FA) synthesis, and selective sensitivity to pharmacological inhibition of FAS activity by cerulenin or the novel compound C75. In this study, inhibition of FA synthesis at the physiologically regulated step of carboxylation of acetyl-CoA to malonyl-CoA by 5-(tetradecyloxy)-2-furoic acid (TOFA) was not cytotoxic to breast cancer cells in clonogenic assays. FAS inhibitors induced a rapid increase in intracellular malonyl-CoA to several fold above control levels, whereas TOFA reduced intracellular malonyl-CoA by 60%. Simultaneous exposure of breast cancer cells to TOFA and an FAS inhibitor resulted in significantly reduced cytotoxicity and apoptosis. Subcutaneous xenografts of MCF7 breast cancer cells in nude mice treated with C75 showed FA synthesis inhibition, apoptosis, and inhibition of tumor growth to less than 1/8 of control volumes, without comparable toxicity in normal tissues. The data suggest that differences in intermediary metabolism render tumor cells susceptible to toxic fluxes in malonyl-CoA, both in vitro and in vivo.

Pharmacological inhibition of fatty acid synthase activity produces both cytostatic and cytotoxic effects modulated by p53.

Fatty acid synthetic metabolism is abnormally elevated in tumor cells, and pharmacological inhibitors of the anabolic enzyme fatty acid synthase (FAS), including the natural product cerulenin and the novel synthetic compound c75, are selective inhibitors of tumor cell growth. We have recently reported that these two FAS inhibitors both produce rapid, potent inhibition of DNA replication and S-phase progression in human cancer cells, as well as apoptotic death. Here we report an additional characterization of the cellular response to FAS inhibition. RKO colon carcinoma cells were selected for study because they undergo little apoptosis within the first 24 h after FAS inhibition. Instead, RKO cells exhibited a biphasic stress response with a transient accumulation in S and G2 at 4 and 8 h that corresponds to a marked reduction in cyclin A- and B1-associated kinase activities, and then by accumulation of p53 and p21 proteins at 16 and 24 h and growth arrest in G1 and G2. The response of RKO cells to FAS inhibition resembled a genotoxic stress response, but DNA damage did not appear to be an important downstream effect of FAS inhibition, because none was detected using the single cell gel electrophoresis assay (comet assay) to assess DNA damage. p53 function is probably important in protecting RKO cells from FAS inhibition because, similar to many other tumor lines, RKO cells expressing a dominant negative mutant p53 gene underwent extensive apoptosis within 24 h after FAS inhibition. Sensitization of cells to FAS inhibitors by the loss of p53 raises the possibility that these agents may be clinically useful against malignancies carrying p53 mutations. Whereas induction of apoptosis appeared related to accumulation of the substrate, malonyl-CoA, after FAS inhibition, the cytostatic effects were independent of malonyl-CoA accumulation and may have resulted from product depletion.

A novel gene expressed in human keratinocytes with long-term in vitro growth potential is required for cell growth.

The herpes simplex virus large subunit of ribonucleotide reductase differs from its counterparts in eukaryotic and prokaryotic cells and in other viruses in that it contains a unique domain that codes for a distinct serine-threonine protein kinase that activates the Ras/MEK/MAPK mitogenic pathway and is required for virus growth. Previous studies suggested that ribonucleotide reductase protein kinase was co-opted from a cellular gene. Cellular genes similar to ribonucleotide reductase protein kinase were not cloned, however, and their function is unknown. Here we report that a novel gene (H11) that codes for a protein similar to herpes simplex virus 2 ribonucleotide reductase protein kinase, is expressed in skin tissues, cultured keratinocytes, and the keratinocyte cell line A431. The protein is phosphorylated and it associates with the plasma membrane. H11 is expressed in keratinocytes with long-term in vitro growth potential and is coexpressed with high levels of adhesion molecules involved in signal transduction, such as beta1 integrin. Antisense oligonucleotides that inhibit H11 expression inhibit DNA synthesis and keratinocyte proliferation, suggesting that H11 expression is required for cell growth.

The origin of red cell fluorescence caused by hydrogen peroxide treatment.

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.

HIV infection and aging: mechanisms to explain the accelerated rate of progression in the older patient.

Age is an important predictor of progression in HIV infections. Not only do older individuals' develop AIDS more rapidly than younger persons, they die more quickly after developing an AIDS-defining illness. While the elderly have higher morbidity and mortality rates from viral and bacterial infections, the mechanism(s) responsible for the more rapid progression of HIV infection in older individuals has not been described. Our results demonstrate that the destruction of T cells in both young and old HIV infected patients progresses at the same rate. HIV 1-infected cells from older individuals do not appear more susceptible to immune mediated destruction. The more rapid progression appears due to an inability of older persons to replace functional T cells that are being destroyed. These findings suggest that improved survival in older HIV infected individuals will require more aggressive antiretroviral therapies as well as continued research to identify and preserve immune system elements that control the virus.

Carboxyfluorescein diacetate labeling does not affect adhesion molecule expression or function in human neutrophils or eosinophils.

Fluorescently labeled leukocytes are commonly used in in vitro and in vivo experimental systems. However, the effects of fluorescent labeling on the expression and function of leukocyte adhesion molecules has not been examined in part because the extreme intensity of fluorescence tends to obscure signals from other fluorochromes used for dual color analysis. We have utilized a novel technique involving a 7-amino-4-methylcoumarin-3-acetic acid (AMCA) fluorophore-conjugated F(ab')2 fragment excitable in the ultraviolet wavelength range (350-450 nm) and dual-laser flow cytometry to determine if labeling of human neutrophils and eosinophils with the fluorescent dye 5-(6)-carboxyfluorescein diacetate (CFDA) alters surface expression of the primary leukocyte adhesion molecules involved in leukocyte-endothelial interactions. Simultaneously, adhesion molecule function was assessed by comparing the ability of CFDA-labeled vs. control cells to adhere to cultured human umbilical vein endothelial cells (HUVEC) and purified immobilized adhesion molecules. Isolated human eosinophils and neutrophils were fluorescently labeled by incubation with CFDA. Flow cytometric comparisons of labeled and unlabeled cells demonstrated that fluorescence labeling of neutrophils and eosinophils with CFDA did not alter basal surface expression of the beta 2 integrins (i.e., CD11a CD11b or, CD18). Stimulation of neutrophils with fMLP and eosinophils with PMA resulted in increased surface expression of CD11b and CD18 which was not altered by CFDA labeling. Likewise, CFDA labeling of neutrophils and eosinophils did not significantly alter their integrin-dependent adhesion to activated HUVEC under static or rotational conditions. Similarly, adhesion to immobilized recombinant E- and P-selectin was unaltered. These data demonstrate that fluorescent labeling of human neutrophils and eosinophils with CFDA does not alter surface expression or function of several adhesion molecules necessary for leukocyte-endothelial interactions. The use of CFDA-labeled cells in experiments employing intravital microscopy should therefore provide valid information on adhesion molecule function in vivo.

Increased production of TGF-beta and Il-6 by aged spleen cells.

Aging is accompanied by a progressive decline in immunity in every species that has been studied. Despite its ubiquity, the causes of immunosenescence are unknown. Transforming growth factor beta (TGF-beta) is a cytokine with potent immunosuppressive properties. Cells from aged mice produce increased levels of TGF-beta in vitro along with similar increases in interleukin 6 (Il-6), a cytokine which is immunosuppressive at elevated concentrations. Il-6 does not upregulate TGF-beta production, but high concentrations of Il-6 increase the percentage of cells expressing the TGF-beta receptor. Increased TGF-beta production and Il-6-induced upregulation of the TGF-beta receptor may be factors contributing to age-associated immunosuppression.

Dopamine stimulates redox-tyrosine kinase signaling and p38 MAPK in activation of astrocytic C6-D2L cells.

An increase in dopamine (DA) availability in rat brain has been suggested to participate in certain neurodegenerative processes. However, the regulatory effects of DA on glial cells have not been extensively studied. Using a rat C6 glioma cell line stably expressing recombinant D2L receptors, we have found that micromolar levels of DA stimulate mitogenesis and glial fibrillary acidic protein (GFAP) expression, both serving as parameters of reactive gliosis. This mitogenesis occurs about 29 h after exposure to DA and requires D2-receptor-mediated intracellular redox-tyrosine kinase activation. Either DA or quinpirole, a D2 receptor agonist, stimulates protein tyrosine phosphorylation. Application of either DPI, a potent inhibitor of NADPH-dependent oxidase, or NAC, an anti-oxidant, effectively prevented DA-induced tyrosine phosphorylation and DNA synthesis. Preincubation of (+)-butaclamol, a D2 receptor antagonist, inhibits both DA-stimulated tyrosine phosphorylation and mitogenesis. DA at micromolar levels also stimulates GFAP expression. This DA-regulated GFAP expression can be completely inhibited by SB203580, a selective p38 MAPK inhibitor, but not influenced by (+)-butaclamol and genistein, a protein tyrosine kinase inhibitor. Thus, our data suggest that regulation of DNA synthesis and GFAP expression induced by DA is mediated by independent signaling pathways. The mitogenesis requires a D2-receptor-mediated protein tyrosine kinase cascade, while GFAP expression needs a D2-receptor-independent p38 MAPK activation. This observation may help to understand the processes of reactive gliosis in some dopaminergic-related neurodegenerative diseases.

Modulation of major histocompatibility complex antigens and inhibition of proliferative activity of YAC lymphoma cells by a natural killer lysis resistance-inducing factor (NK-LRIF).

Concanavalin-A-activated rat spleen cells secrete a natural killer lysis resistance-inducing factor (NK-LRIF) distinct from interleukin-2 and interferon, which induces resistance to NK-cell-mediated lysis in YAC tumor cells. In order for NK-LRIF to have an effect on YAC cells, several hours of incubation is required. When NK-LRIF-treated YAC cells are washed and cultured in the absence of NK-LRIF, normal NK susceptibility is regained. YAC cells treated with NK-LRIF show a significant decrease in the rate of proliferation as judged by changes in cell numbers and rate of thymidine incorporation. Cell cycle studies indicate that the proportion of G0/G1 phase cells increases in YAC preparations treated with NK-LRIF. Major histocompatibility complex (MHC) class I antigen expression is markedly enhanced on YAC cells incubated with NK-LRIF but the expression of MHC class II antigens and Thy-1 antigen remains unchanged. No effect of NK-LRIF treatment on the capacity of YAC cells to bind effector spleen cells could be demonstrated.

Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.

Mitogenic activity of 12-O-tetradecanoyl phorbol-13-acetate on peripheral blood lymphocytes from young and aged adults.

The effect of age on the proliferative response to 12-O-tetradecanoyl phorbol-13-acetate (TPA) was examined using peripheral blood lymphocytes from 185 adults. TPA-induced DNA synthesis measured by cellular 3H-thymidine incorporation was found, like the responses of cells activated by PHA and Con A, to markedly diminish with advancing age. The presence of indomethacin (1 microgram/ml) or Ro 20-5720 (10 micrograms/ml) in TPA activated cell cultures, unlike PHA stimulated cultures, did not result in augmentation of 3H-thymidine incorporation by cells from elderly individuals. These results demonstrate that prostaglandin synthesizing suppressor cells are not responsible for the age-related depression of cellular immune function observed in TPA activated cells and confirm the observation that decreased production and/or utilization of soluble mediators, such as IL-2, may account for the diminished mitogen responsiveness of lymphocytes from elderly individuals.

Enumeration of T lymphocyte subsets by monoclonal antibodies in young and aged humans.

The percentage and absolute number of peripheral blood mononuclear cells reactive with monoclonal antibodies identifying mature thymocytes and T cells (T3+), helper/inducer cells (T4+), and suppressor/cytotoxic cells (T8+) was determined in 19 young (mean age 35 yr) and 31 elderly (mean age 72 yr) individuals. The percent representation but not the absolute number of T cells (T3+) declined significantly (p less than 0.001) in the elderly, and the decline was attributable to both an absolute and relative decrease in the representation of the subpopulation of cytotoxic/suppressor (T8+) cells. The percentage and number of helper/inducer (T4+) T cells was comparable in both age groups.

Phenotypic expression of natural killer cell associated membrane antigens and cytolytic function of peripheral blood cells from different aged humans.

The function and phenotypes of peripheral blood NK cells from different aged adults were examined using a 4 hr 51Cr release NK assay and laser flow cytometry utilizing the Leu-7 and Leu-11 monoclonal antibodies. It was found that there was no difference between aged and young adults in the expression of Leu-7+, Leu-11+, Leu-7-11+ or Leu-7+11+ cells. A significant decrease, however, was seen in both percent representation and absolute number of Leu-7+11- cells in older subjects. When examined by age and gender, women aged 17-59 yr had a significantly lower percent representation and absolute number of Leu-11+, Leu-7-11+ and Leu-7+11+ cells and baseline NK activity than did men of similar age. No differences were seen between men and women aged 60-84 yr. No differences were seen in the degree of augmentation by interleukin-2 of NK function in any group. Our data indicate that NK function is correlated with cells bearing the Leu-11 surface antigen and that the gender of the cell donor is a significant factor in the representation of NK cells and function in persons under 60 yr of age. Studies on age differences in NK number and function must therefore take into account the percent of women in the populations studied.

Impaired phorbol ester and calcium ionophore induced proliferation of T cells from old humans.

Current models of T cell activation implicate increases in intracellular free Ca2+ concentration and activation of the Ca2+ and phospholipid dependent enzyme protein kinase C (PKC) as important early events leading to interleukin 2 (IL-2) production, interleukin 2 receptor (IL-2R) expression, and subsequent cell proliferation. The present study examined the age-related defect in T cell proliferation to determine if signals that activate PKC and increase intracytosolic free Ca2+ concentration might be defective. Using phorbol myristate acetate (PMA), which directly activates PKC, and Ca2+ ionophore A23187, which increases intracellular cytoplasmic free Ca2+ concentration, the induction of IL-2 secretion, IL-2R expression and cell proliferation were studied. The results demonstrate that following stimulation with PMA and A23187, purified T cells from elderly subjects demonstrate low levels of IL-2 production, IL-2R expression and cell proliferation. Exogenous purified human IL-2 did not fully correct the low proliferative responses of T cells from old donors, however, did markedly boost the response. While it appears that the inability of T cells to express IL-2R and respond to IL-2, along with a lower endogenous IL-2 production are limiting factors in cell proliferation, the inability of PMA and A23187 to correct this defect suggests that the early phases of signal transduction per se are probably not a primary cause of the immunodeficiency seen in ageing.