The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood–brain barrier disruption: Implications for multiple sclerosis treatment

Experimental autoimmune encephalomyelitis (EAE) is a T cell autoimmune disorder that is a widely used animal model for multiple sclerosis (MS) and, as in MS, clinical signs of EAE are associated with blood–brain barrier (BBB) disruption. SR 57746A, a nonpeptide drug without classical immunosuppressive properties, efficiently protected the BBB and impaired intrathecal IgG synthesis (two conventional markers of MS exacerbation) and consequently suppressed EAE clinical signs. This compound inhibited EAE-induced spinal cord mononuclear cell invasion and normalized tumor necrosis factor α and IFN-γ mRNA expression within the spinal cord. These data suggested that pharmacological intervention aimed at inhibiting proinflammatory cytokine expression within the central nervous system provided protection against BBB disruption, the first clinical sign of EAE and probably the key point of acute MS attacks. This finding could lead to the development of a new class of compounds for oral therapy of MS, as a supplement to immunosuppressive agents.

Identification of a Saccharomyces cerevisiae Gene that Is Required for G1 Arrest in Response to the Lipid Oxidation Product Linoleic Acid Hydroperoxide*

Reactive oxygen species cause damage to all of the major cellular constituents, including peroxidation of lipids. Previous studies have revealed that oxidative stress, including exposure to oxidation products, affects the progression of cells through the cell division cycle. This study examined the effect of linoleic acid hydroperoxide, a lipid peroxidation product, on the yeast cell cycle. Treatment with this peroxide led to accumulation of unbudded cells in asynchronous populations, together with a budding and replication delay in synchronous ones. This observed modulation of G1 progression could be distinguished from the lethal effects of the treatment and may have been due to a checkpoint mechanism, analogous to that known to be involved in effecting cell cycle arrest in response to DNA damage. By examining several mutants sensitive to linoleic acid hydroperoxide, the YNL099c open reading frame was found to be required for the arrest. This gene (designated OCA1) encodes a putative protein tyrosine phosphatase of previously unknown function. Cells lacking OCA1 did not accumulate in G1 on treatment with linoleic acid hydroperoxide, nor did they show a budding, replication, or Start delay in synchronous cultures. Although not essential for adaptation or immediate cellular survival, OCA1 was required for growth in the presence of linoleic acid hydroperoxide, thus indicating that it may function in linking growth, stress responses, and the cell cycle. Identification of OCA1 establishes cell cycle arrest as an actively regulated response to oxidative stress and will enable further elucidation of oxidative stress-responsive signaling pathways in yeast.

Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA

dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P.

A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle

ATP-binding cassette (ABC) transporters bind and hydrolyze ATP. In the cystic fibrosis transmembrane conductance regulator Cl− channel, this interaction with ATP generates a gating cycle between a closed (C) and two open (O1 and O2) conformations. To understand better how ATP controls channel activity, we examined gating transitions from the C to the O1 and O2 states and from these open states to the C conformation. We made three main observations. First, we found that the channel can open into either the O1 or O2 state, that the frequency of transitions to both states was increased by ATP concentration, and that ATP increased the relative proportion of openings into O1 vs. O2. These results indicate that ATP can interact with the closed state to open the channel in at least two ways, which may involve binding to nucleotide-binding domains (NBDs) NBD1 and NBD2. Second, ATP prolonged the burst duration and altered the way in which the channel closed. These data suggest that ATP also interacts with the open channel. Third, the channel showed runs of specific types of open–closed transitions. This finding suggests a mechanism with more than one cycle of gating transitions. These data suggest models to explain how ATP influences conformational transitions in cystic fibrosis transmembrane conductance regulator and perhaps other ABC transporters.

The treatment of chronic progressive multiple sclerosis with cladribine.

A 2-year, placebo-controlled, double-blind, crossover study was started in 1992 to evaluate cladribine, an immunosuppressive drug, in the treatment of chronic progressive multiple sclerosis. In the first year patients were given cladribine 0.10 mg/kg per day for 7 days as four monthly courses for a total of 2.8 mg/kg or placebo. During the second year patients treated with placebo during the first year were given i.v. infusions of 0.10 mg, 0.05 mg, and 0.05 mg of cladribine per kg of body weight per day for 7 consecutive days in three successive monthly courses, for a total dose of 1.4 mg/kg. Patients who had been treated previously with cladribine were crossed over to placebo. Analysis of the results revealed a favorable influence on the neurological performance scores, both in the Kurtze extended disability status and the Scripps neurological rating scale, and on MRI findings in patients treated with cladribine. In the first year the most striking finding was that while clinical deterioration continued in the placebo-treated patients, the condition of patients who received cladribine stabilized or even improved slightly. Toxicity and therapeutic response were dose-related.

RB-mediated suppression of spontaneous multiple neuroendocrine neoplasia and lung metastases in Rb+/− mice

Alterations in pathways mediated by retinoblastoma susceptibility gene (RB) product are among the most common in human cancer. Mice with a single copy of the Rb gene are shown to develop a syndrome of multiple neuroendocrine neoplasia. The earliest Rb-deficient atypical cells were identified in the intermediate and anterior lobes of the pituitary, the thyroid and parathyroid glands, and the adrenal medulla within the first 3 months of postnatal development. These cells form gross tumors with various degrees of malignancy by postnatal day 350. By age of 380 days, 84% of Rb+/− mice exhibited lung metastases from C-cell thyroid carcinomas. Expression of a human RB transgene in the Rb+/− mice suppressed carcinogenesis in all tissues studied. Of particular clinical relevance, the frequency of lung metastases also was reduced to 12% in Rb+/− mice by repeated i.v. administration of lipid-entrapped, polycation-condensed RB complementary DNA. Thus, in spite of long latency periods during which secondary alterations can accumulate, the initial loss of Rb function remains essential for tumor progression in multiple types of neuroendocrine cells. Restoration of RB function in humans may prove an effective general approach to the treatment of RB-deficient disseminated tumors.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Activity and nature of p21WAF1 complexes during the cell cycle

Elevated levels of the p21WAF1 (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins (“free” p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21.

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: A possible mechanism of breast carcinogenesis

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Evaluating treatment protocols to prevent antibiotic resistance

The spread of bacteria resistant to antimicrobial agents calls for population-wide treatment strategies to delay or reverse the trend toward antibiotic resistance. Here we propose new criteria for the evaluation of the population-wide effects of treatment protocols for directly transmitted bacterial infections and discuss different usage patterns for single and multiple antibiotic therapy. A mathematical model suggests that the long-term benefit of single drug treatment from introduction of the antibiotic until a high frequency of resistance precludes its use is almost independent of the pattern of antibiotic use. When more than one antibiotic is employed, sequential use of different antibiotics in the population (“cycling”) is always inferior to treatment strategies where, at any given time, equal fractions of the population receive different antibiotics. However, treatment of all patients with a combination of antibiotics is in most cases the optimal treatment strategy.

Potent inhibition of tumor survival in vivo by β-lapachone plus taxol: Combining drugs imposes different artificial checkpoints

Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of β-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after β-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. β-Lapachone causes cell-cycle delays in late G1 and S phase, and taxol arrests cells at G2/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone “collisions” at cell-cycle checkpoints.

Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

Anti-CD14 mAb treatment provides therapeutic benefit after in vivo exposure to endotoxin

The presence of endotoxin from Gram-negative bacteria signals the innate immune system to up-regulate bacterial clearance and/or killing mechanisms. Paradoxically, such responses also contribute to septic shock, a clinical problem occurring with high frequency in Gram-negative septicemia. CD14 is a receptor for endotoxin (lipopolysaccharide, LPS) and is thought to have an essential role in innate immune responses to infection and thereby in the development of septic shock. Using a novel rabbit model of endotoxic shock produced by multiple exposures to endotoxin, we show that anti-rabbit CD14 mAb, which blocks LPS-CD14 binding, protects against organ injury and death even when the antibody is administered after initial exposures to LPS. In contrast, anti-rabbit tumor necrosis factor mAb treatment fails to protect when administered after LPS injections. These results support the concept that anti-CD14 treatment provides a new therapeutic window for the prevention of pathophysiologic changes that result from cumulative exposures to LPS during septic shock in man.