THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the Ig chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.

Continuous cultures of fused cells secreting antibody of predefined specificity

It is proposed that most neoplasms arise from a single cell of origin, and tumor progression results from acquired genetic variability within the original clone allowing sequential selection of more aggressive sublines. Tumor cell populations are apparently more genetically unstable than normal cells, perhaps from activation of specific gene loci in the neoplasm, continued presence of carcinogen, or even nutritional deficiencies within the tumor. The acquired genetic insta0ility and associated selection process, most readily recognized cytogenetically, results in advanced human malignancies being highly individual karyotypically and biologically. Hence, each patient's cancer may require individual specific therapy, and even this may be thwarted by emergence of a genetically variant subline resistant to the treatment. More research should be directed toward understanding and controlling the evolutionary process in tumors before it reaches the late stage usually seen in clinical cancer.

http://science.sciencemag.org/content/sci/194/4260/23.full.pdf

The clonal evolution of tumor cell populations

A search of a 35-kilobase region of the human Y chromosome necessary for male sex determination has resulted in the identification of a new gene. This gene is conserved and Y-specific among a wide range of mammals, and encodes a testis-specific transcript. It shares homology with the mating-type protein, Mc, from the fission yeast Schizosaccharomyces pombe and a conserved DNA-binding motif present in the nuclear high-mobility-group proteins HMG1 and HMG2. This gene has been termed SRY (for sex-determining region Y) and proposed to be a candidate for the elusive testis-determining gene, TDF.

A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif

ion. The oxidative addition mechanism was originally proposed22i because of the lack of a strong rate dependence on polar factors and on the acidity of the medium. Later, however, the electrophilic substitution mechanism also was proposed. Recently, the oxidative addition mechanism was confirmed by investigations into the decomposition and protonolysis of alkylplatinum complexes, which are the reverse of alkane activation. There are two routes which operate in the decomposition of the dimethylplatinum(IV) complex Cs2Pt(CH3)2Cl4. The first route leads to chloride-induced reductive elimination and produces methyl chloride and methane. The second route leads to the formation of ethane. There is strong kinetic evidence that the ethane is produced by the decomposition of an ethylhydridoplatinum(IV) complex formed from the initial dimethylplatinum(IV) complex. In D2O-DCl, the ethane which is formed contains several D atoms and has practically the same multiple exchange parameter and distribution as does an ethane which has undergone platinum(II)-catalyzed H-D exchange with D2O. Moreover, ethyl chloride is formed competitively with H-D exchange in the presence of platinum(IV). From the principle of microscopic reversibility it follows that the same ethylhydridoplatinum(IV) complex is the intermediate in the reaction of ethane with platinum(II). Important results were obtained by Labinger and Bercaw62c in the investigation of the protonolysis mechanism of several alkylplatinum(II) complexes at low temperatures. These reactions are important because they could model the microscopic reverse of C-H activation by platinum(II) complexes. Alkylhydridoplatinum(IV) complexes were observed as intermediates in certain cases, such as when the complex (tmeda)Pt(CH2Ph)Cl or (tmeda)PtMe2 (tmeda ) N,N,N′,N′-tetramethylenediamine) was treated with HCl in CD2Cl2 or CD3OD, respectively. In some cases H-D exchange took place between the methyl groups on platinum and the, CD3OD prior to methane loss. On the basis of the kinetic results, a common mechanism was proposed to operate in all the reactions: (1) protonation of Pt(II) to generate an alkylhydridoplatinum(IV) intermediate, (2) dissociation of solvent or chloride to generate a cationic, fivecoordinate platinum(IV) species, (3) reductive C-H bond formation, producing a platinum(II) alkane σ-complex, and (4) loss of the alkane either through an associative or dissociative substitution pathway. These results implicate the presence of both alkane σ-complexes and alkylhydridoplatinum(IV) complexes as intermediates in the Pt(II)-induced C-H activation reactions. Thus, the first step in the alkane activation reaction is formation of a σ-complex with the alkane, which then undergoes oxidative addition to produce an alkylhydrido complex. Reversible interconversion of these intermediates, together with reversible deprotonation of the alkylhydridoplatinum(IV) complexes, leads to multiple H-D exchange

Activation of c-h bonds by metal complexes

For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of “the dose makes the poison,” because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from...

/pdf/hormones-and-endocrine-disrupting-chemicals-low-dose-effects-2vn32zoqc8.pdf

Hormones and endocrine-disrupting chemicals: Low-dose effects and nonmonotonic dose responses

Malignancy can be suppressed when malignant cells are fused with certain non-malignant ones. The hybrid cells derived from such fusions give rise to segregants in which a loss of chromosomes is associated with reversion to malignancy. The expression of histo-compatibility antigens can also be suppressed when cells bearing these antigens are fused with others that express them poorly.

Suppression of Malignancy by Cell Fusion

Hybrid Cells Derived from Mouse and Man : Artificial Heterokaryons of Mammalian Cells from Different Species

Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species.

If human cells are subjected to large doses of ionising radiation and then fused with rodent cells, hybrid clones are obtained in which linked human genes may be segregated. By measuring the frequency with which pairs of linked genes are cotransferred after irradiation, it is possible to determine the linear order of groups of genes and to estimate the distances between them. This technique offers a new and systematic approach to the mapping problem.

New method for mapping genes in human chromosomes.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.

A virus, inactivated by ultraviolet light, was used to fuse together cells from different species of vertebrate, and the resulting heterokaryons were examined by autoradiographic and cytological techniques. Heterokaryons could be made with both differentiated and undifferentiated cells: HeLa and Ehrlich ascites cells were studied as examples of undifferentiated cells; rabbit macrophages, rat lymphocytes and hen erythrocytes as examples of differentiated cells. These last three cells were chosen because in them, in varying degrees, the process of differentiation has resulted in suppression of the synthesis of DNA or of both DNA and RNA. This suppression was in all cases found to be reversible: the dormant nuclei could be induced to resume the synthesis of RNA or DNA or both when the differentiated cells were fused with a cell which normally synthesizes RNA and DNA. Observations on heterokaryons in which differentiated cells were fused with HeLa cells and with each other permitted certain general conclusions to be drawn about the regulation of nucleic acid synthesis in the heterokaryon. It was found that if either one of the parent cells normally synthesized RNA, RNA synthesis took place in both types of nuclei in the heterokaryon. If either of the parent cells normally synthesized DNA, DNA synthesis took place in both types of nuclei in the heterokaryon. If neither of the parent cells synthesized DNA, no DNA synthesis took place in the heterokaryon. In all cases where a cell which synthesized a particular nucleic acid was fused with one which did not, the active cell initiated the synthesis of this nucleic acid in the inactive partner. In no case did the inactive cell suppress synthesis in the active partner. The nuclei of heterokaryons in which DNA synthesis took place underwent mitosis, and those nuclei which entered mitosis synchronously usually fused together. This process resulted in the progressive formation of mononucleate hybrid cells, which might thus contain within a single nucleus chromosomal complements derived from different species. These mononucleate hybrid cells were also capable of RNA and DNA synthesis, and many of them in turn underwent mitosis. At metaphase these cells showed, in various combinations, the chromosomal complements of the two parent cells. Mononucleate hybrid cells formed by the fusion of a large number of single cells did not appear to be capable of continued multiplication; but mononucleate cells containing one chromosomal set from each parent cell were still found to be undergoing mitosis many days after cell fusion.

/pdf/artificial-heterokaryons-of-animal-cells-from-different-2p9wrzm83d.pdf

Henry Harris

Papers

Suppression of Malignancy by Cell Fusion

Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species.

New method for mapping genes in human chromosomes.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.

Artificial heterokaryons of animal cells from different species.