Genetic control of DNA breakdown and repair inE. coli K-12 treated with mitomycin C or ultraviolet light

Summary Ultraviolet light sensitive mutants ofE. coli defective at theuvrA,uvrB oruvrC locus showed increased sensitivity to the lethal effects of mitomycin C when compared with theuvr ⁺ parental strain. In addition, DNA breakdown after treatment of cells with either mitomycin C or with ultraviolet light was greater in the parental strain carrying the activeuvr ⁺ genes than inuvr mutants. Thus, injuries produced by either mitomycin C or by ultraviolet light may be repaired by the same molecular mechanism which has been proposed and which involves defect excision, single strand breakdown and reconstruction of the DNA.With 2 Figures in the Text     

Interaction of peritoneal exudate lymphocytes with histocompatibility antigens.

The effect of allo-immunization on the response of mouse peritoneal exudate lymphocytes (PELs) in mixed lymphocyte culture (MLC) and cell mediated cytotoxicity was described. In characterizing these responses, the results were compared with data obtained in similar experiments with splenic lymphocytes (SpLs). While unimmunized BALB/c SpLs (H-2^d) showed strong reactivity in the one way MLC against irradiated C57BL/6 spleen cells, unimmunized PELs gave a barely detectable response. Subcutaneous (sc) but not intravenous (iv) allo-immunization resulted in a transient but marked increase in MLC reactivity by PELs. Immunization by either route resulted in an augmented MLC by SpLs. Further, sc, but not iv, allo-immunization resulted in the transient appearance of cellular cytotoxic lymphocytes in PELs. It was concluded that PELs were unique among secondary lymphoid populations in that they contained very few histocompatibility antigen reactive cells in the absence of immunization; and as such represented a population committed solely to antigens which the animal had recently experienced.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Selective proliferation of T cells in the mixed lymphocyte interaction

The contribution of avian thymus-derived (T) and bursa-derived (B₂) cells to the proliferating cell population in mixed lymphocyte cultures (MLC) was evaluated. When spleen cells of chickens containing chromosomally marked T and B₂ cells were stimulated in a one-way MLC by mitomycin C blocked allogeneic spleen cells, only T cells proliferated during a 4–9 day culture period. No evidence for significant recruitment of B₂ cells, expressed as proliferation of B₂ cells, was found. The initial viability and proliferative potential of B₂ cells was shown by a substantial and selective B₂ cell response to anti-immunoglobulin serum.     

Cytogenetics and the major histocompatibility complex in a proliferating microcell-mediated hybrid clone

Summary By way of a microcell fusion, three chromosomes from a B82HTQ2 (TK^–) cell were introduced into a PG19 (HGPRT^–) cell. Analysis of this hybrid clone showed that the transferred chromosomes restored a positive HGPRT status but failed to produce heterozygosity for the major histocompatibility complex (H-2). The three chromosomes also proved stable in both long term culture in vitro and tumor testing in mice. It is suggested that the method could prove useful in correcting genetic defects or in introducing new genetic characteristics without the introduction of the genes coding for major histocompatibility antigens. The surface structure of the microcells was studied by scanning electron microscope. The optimum for induction of the microcells from B82HTQ2 cells and its' purification were reported here. Frequency of the sister chromatid exchange (SCE) of the hybrid cells and their sensitivity to mitomycin C (MMC) were also examined.     

Specificity of human histocompatibility antigen reactive cells in vitro. Separate cell populations responsive to the products of each major histocompatibility system (MHS) haplotype

One-way mixed lymphocyte cultures were established between related cell donors A (haplotype designated $\text{a}\text{b}$) and B ($\text{a}\text{c}$). The cells from A, proliferating in response to stimulation by mitomycin treated cells from B, were eliminated from the culture by a hot pulse of ³H-thymidine. A marginal response was observed when the remaining cells from A reencountered additional stimulating cells from B, or cells from an HL-A identical sibling to B. In addition, the remaining responding cells were virtually incapable of responding to secondary stimulation by family member C ($\text{b}\text{c}$), who shared one haplotype (b) with individual A and the other haplotype (c) with the individual stimulating cell donor B. The MLC secondary stimulation response to family member D ($\text{c}\text{d}$), who differed from A by both haplotypes, but shared one haplotype with B, was reduced to approximately 50% of control values. In other experiments it was found possible to completely eliminate the response of A ($\text{a}\text{b}$) to D ($\text{c}\text{d}$) by using a combination of stimulating cells from related donors B ($\text{a}\text{b}$) and C ($\text{b}\text{c}$) in the initial hot pulse MLC.Separate populations of responding cells reactive to antigenic products of each major histocompatibility system haplotype is a likely explanation of these observations.     

The expression of Mls c determinants on Mls a,Mls b,and Mls x prototypic strains

In the mouse sytem, specific determinants other than major histocompatibility complex (MHC) gene products are capable of inducing strong primary proliferative responses in naive T cells. These determinants are encoded by at least two gene loci designated as minor lymphocyte stimulatory (Mls) loci. In order to elucidate the biological role of the Mls system, an effort has been initiated to clarify the fundamental immunogenetic characteristics of the Mls system. In this report, we describe the unexpected finding that Mls ^c determinants are expressed on splenocytes of strains including those which have been used as prototypic examples of three other Mls types: Mls ^a (DBA/2, DBA/1), Mls ^b , (BALB/c), and Mls ^x (PL/J). The expression of Mls ^c by these strains was demonstrated both by the response patterns of unprimed T cells from MHC-identical inbred or F₁ hybrid strains and by the responses of a panel of Mls-specific T-cell clones. The experimental results reported here also suggest that the expression of Mls determinants may be influenced by multiple other genes, including MHC-linked genes.Abbreviations used in this paper MHC major histocompatibility complex - MLR mixed lymphocyte reaction - Mls minor lymphocyte stimulating locus antigen - MMC mitomycin C - NNT nylon wool nonadherent T cells     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells. I. Culture conditions resulting in augmented cell-mediated cytotoxicity

Culture conditions have been established that result in the sensitization of normal human peripheral blood lymphoid cells on allogeneic melanoma monolayers. Optimal culture conditions require 2 to 8 × 10⁶ mitomycin C treated stimulator melanoma cells to sensitize 5 to 10 × 10⁶ responder lymphoid cells. Neither rocking nor refeeding of the culture is necessary for the sensitization procedure. Stimulator cells grown in either fetal calf serum or human serum will serve as effective stimulator or target cells. Peak cytotoxic activity was detected at 44 hr in a microcytotoxicity assay, although some cytotoxic activity was detectable at 24 hr.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Potentiation of bleomycin cytotoxicity by membrane-interacting drugs and increased calcium ions

The cytotoxic effect of the antitumor antibiotic peplomycin (PEP), a new member of bleomycin group antibiotics, toward HeLa cells and mouse FM3A cells is enhanced by some membrane-interacting drugs such as verapamil, persantin, prenylamine, chlorpromazine and anafranil. The enhancing action of verapamil is selective to this group antibiotics, since it does not potentiate the cytotoxic effects of vincristine, adriamycin, mitomycin C, cis-diamminedichloroplatinum(II) and macromomycin. An enhanced PEP cytotoxicity has been also demonstrated by the treatment of cells in the presence of increased CaCl₂. This enhancing effect of increased CaCl₂ is prevented by the Ca²⁺ transport inhibitor ruthenium red. Since these membrane-directed drugs have been shown to affect Ca²⁺ metabolism, we conclude that potentiation of PEP cytotoxicity by these drugs is mediated by an increase in intracellular Ca²⁺.     

In vitro tumor cell killing by peritoneal macrophages from mitomycin C-treated rats

Summary The local cellular response induced by intraperitoneal injection of mitomycin C was examined in terms of cell-mediated cytotoxicity for tumor cells. An in vitro cytolysis assay involving ¹²⁵I-iododeoxyuridine-labeled tumor target cells revealed that treatment of normal ACI/N rats (200 g) with a single intraperitoneal injection of mitomycin C (50, 100, or 200 g) induced tumoricidal macrophages in the peritoneal cavity. The tumoricidal activity was dependent on the dose of mitomycin C injected and it was detectable as early as 1 day after the intraperitoneal injection of mitomycin C. In addition to the increased tumoricidal activity, the functional activities of the peritoneal macrophages were found to be increased with respect both to uptake of 2-deoxy-d-glucose and to phagocytosis of latex beads. Additional experiments excluded the possibility that the tumor cell cytolysis was the result of direct cytotoxicity by mitomycin C that might have been incorporated in the peritoneal macrophages or of nutrient depletion in the medium during the cytolysis assay. Furthermore, endotoxin contamination of the mitomycin C, which might have produced the activated macrophages, was not detected. The mechanism by which mitomycin C injected intraperitoneally induced the tumoricidal macrophages locally remains uncertain; however, it is possible also in clinical situations.     

HLA class l specific t lymphocyte clones with dual alloreactive functions

Four human T lymphocyte clones exhibiting proliferative responses to class I HLA antigens were isolated from an in vitro mixed lymphocyte culture (MLC). Three clones expressed the Leu-2⁺3^– phenotype and demonstrated proliferation in response to HLA-B8, while the fourth clone expressed the Leu-2^–3⁺ phenotype and proliferated in response to HLA-A2. These clones were also cytotoxic towards cells bearing the same target antigens. Blocking studies utilizing monoclonal antibodies demonstrated that proliferation was triggered by determinants on the class I molecule itself, and these determinants appear to be spatially close to those which determine serologic allospecificity. These findings support the concept that the class I molecules themselves are the weak MLC stimulating determinants previously mapped to the HLA-A and B regions of the major histocompatibility complex.Abbreviations used in this paper B-LCL B-lymphoblastoid cell line - cpm counts per minute - FCS fetal calf serum - HS human serum - ³H-TdR tritiated thymidine - IL-2 interleukin-2 - IL2-CM interleukin-2 containing conditioned medium - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MoAb monoclonal antibody - PBL peripheral blood mononuclear leukocytes - PLT primed lymphocyte test     

Alloreactive T-cell clones. Ly phenotypes predict both function and specificity for major histocompatibility complex products

We have studied the association of Ly phenotype with function and specificity for major histocompatibility complex (MHC) products by examining the properties of 21 T-cell clones derived from B10 anti-B 10.D2 and B10.A anti-B10.D2 mixed lymphocyte cultures (MLC). T cells were selected after MLC solely on the basis of Ly phenotype, cloned by limiting dilution, and tested for stability of Ly phenotype, function and specificity for class I or class II MHC products. Sixteen Ly-1⁺2^– and five Ly-1^–2⁺ T-cell clones were tested. The clones selected for the Ly-1 ⁺2^– phenotype maintained this phenotype, expressed helper but not lytic function, and recognized class II MHC products (I-A^d or I-Ed). All Ly-1^–2^– clones maintained this phenotype, possessed cytolytic but not helper activity, and recognized class I MHC products (D^d and L^d). Our data therefore confirm at the clonal level the original observations of a remarkably consistent correlation between Ly markers, MHC specificity, and. function. They suggest that the expression of Ly antigens on T-cell clones forms part of a genetic program for each of these specialized cells that also determines their function and MHC specificity.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - TCGF T cell growth factor (Interleukin-2) - Con A Concanavalin A - DME Dulbecco's modified Eagle's medium - PHA phytohaemagglutinin - LPS lipopolysaccharide - TRF-C T cell replacing factor required for induction of cytolytic cells from thymocytes - PBS phosphate-buffered saline (pH 7.4)     

Expression of the SOS system in Escherichia coli growing under nitrate respiration conditions

Induction of several SOS functions by mitomycin C, bleomycin or thermal treatment of a recA441 mutant growing under nitrate respiration conditions was studied in Escherichia coli. Mitomycin C caused inhibition of cell division, induction of prophages and expression of umuC gene but like in aerobically growing cells, it did not trigger the cessation of cell repiration. On the contrary, both recA⁺ and recA441 cultures either treated with bleomycin or incubated at 42°C failed to induce any of the different SOS functions cited above.Furthermore, after bleomycin addition or thermal treatment both recA⁺ and recA441 cultures did not present any variation in the cellular ATP level, contrary to what happens under aerobic growth. The blocking of the expression of some SOS functions under nitrate respiration conditions is not an irreversible process because cells incubated under these anaerobic conditions were able to induce the SOS system when changed to an aerobic medium 30 min after the SOS-inducing treatment had been applied.     

A search for allelic recombination in Chinese hamster cell hybrids

Summary Mutants resistant to 6-thioguanine were selected from CHO cells which were either temperature sensitive or proline requiring. These mutants were stable and had low levels of hypoxanthine guanine phosphoribosyl transferase (HGPRT). Hybrids were selected which were heteroallelic at the hgprt locus and complementation between the mutants used was not observed. Interallelic recombination at this locus would generate hgprt ⁺ cells which could be selected in Littlefield's HAT medium. Selection experiments with hybrids containing three different pairs of mutants yielded no recombinants among populations of 4x10⁶-2x10⁷ cells. After treatment with the recombinagen mitomycin C, 3 putative recombinants were detected amongst 1.4x10⁷ surviving cells from one hybrid. One of these strains was examined and shown to have a normal level of HGPRT and its heterozygosity at this locus was demonstrated by the segregation of colonies resistant to 6-thioguanine. It cannot be excluded that the rare hgprt ⁺ colonies seen arose by mutation rather than by recombination. Mitotic allelic recombination therefore appears to be a much less frequent event in CHO cells than it is in lower eukaryotes. It is possible that mitotic recombination is effectively suppressed in mammalian cells to prevent the expression of deleterious recessive mutants.     

Synthesis of a major mitomycin C DNA adduct via a triaminomitosene

We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O⁶-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N² and O⁶ positions.     

Anti-θ Serum-indueed Suppression of the Cellular Transfer of Tumour-specific Immunity to a Syngeneic Plasma Cell Tumour

IT has been well documented that tumour-bearing mice can become resistant to their own tumours, especially with chemically induced fibrosarcomas^1–3 and the importance of cell-mediated immune responses rather than humoral antibody in the resistance to tumour transplants has been emphasized^3,4, although the exact mechanism of tumour cell destruction remains ill-defined. Studies in mice^5,6, using allogeneic tumour cells, have demonstrated that thymus-derived (T) lymphocytes are essential for the killing of tumour cells. In addition, using an in vitro method of immunization against histocompatibility antigens, tumour cell destruction either in vitro¹ or in vivo⁸ was shown to be due to T cells alone. In all of these latter studies, however, it is the strong H-2 histocompatibility antigens that are inducing the immune response and not the tumour-specific transplantation antigens (TSTA). We describe here a specific anti-TSTA response to a murine plasma cell tumour which can be transferred with lymphoid cells and which can be shown to involve the essential participation of T cells.     

Biological effects of activation of human peripheral blood mononuclear cells by mitogenic oxidizing agents. I Alloantigen-independent activation of alloimmune memory cells

Mechanisms of activation of alloimmune memory cells by immunologically nonspecific signals were investigated utilizing the mitogenic oxidizing agents, neuraminidase and galactose oxidase (NAGO) and sodium periodate (IO₄^?). Direct activation of primary long-term human mixed-lymphocyte culture (MLC) cells (memory cells) with either NAGO or IO₄^? resulted in increased specific secondary cytolytic activity. Kinetics of the proliferative and cytotoxic responses resulting from such treatment of memory cells paralleled those resulting from treatment of memory cells with irradiated cells that were the stimulators in the primary MLC. Indirect activation of memory cells by NAGO or IO₄^?-treated and irradiated syngeneic cells also resulted in increased specific secondary cytolytic activity. In contrast, peripheral blood mononuclear cells (PBM) activated by the mitogenic oxidizing agents did not develop cytolytic activity toward syngeneic or multiple allogeneic target cells, despite extensive proliferative responses. Cytotoxicity generated by either direct or indirect activation of memory cells by IO₄^? was prevented by treatment of the oxidized cells with the reducing agent, sodium borohydride. Incubation of memory cells in supernatants from 24-hr cultures of PBM activated with either NAGO or IO₄^? resulted in proliferation and in an increase in cytolytic activity in memory cells, but not in PBM. These findings indicate that alloimmune memory cells can be activated by immunologically nonspecific lymphocyte-derived signals that do not depend on alloantigen or lectin.     

β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar¹,Ile⁴,Ile⁸]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.