Lih-Syng Lee

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk−, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk+ clones with a frequency of 10−6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk+ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk− phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10−3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.

Membrane and other Biochemical Effects of the Phorbol Esters and their Relevance to Tumor Promotion

The pleiotropic effects of TPA and related phorbol esters on a variety of cell cultures provide important clues to the process of tumor promotion and the multistep nature of carcinogenesis. These effects can be divided into three categories: 1) mimicry of transformation in normal cells, and enhancement of transformation by chemical carcinogens or oncogenic viruses; 2) modulation (inhibition or induction) of differentiation; and 3) membrane and receptor effects. Recent evidence suggests that TPA acts by binding to specific high affinity cell surface membrane receptors and that this then leads to rapid alterations in the composition of membrane phospholipids. Presumably, these changes in the lipid matrix of cell membranes produce signals or mediators which lead to the subsequent cytoplasmic and nuclear effects of TPA. Thus, whereas the critical target in the action of initiating carcinogens appears to be cellular DNA, the critical target of the phorbol ester tumor promoters appears to be cell membranes. As a unifying concept of two-stage carcinogenesis, we postulate that during the initiation phase in carcinogenesis the covalent binding of carcinogens to DNA induces a host response somewhat analogous to that of the SOS response in bacteria. However, in mammalian cells this response results in abberations in the commitment of the target cells. This may involve ordered events, for example, gene transpositions, rather than random point mutations. Tumor promoters, via their effects on growth, gene expression and differentiation, enhance the selective outgrowth of Initiated cells and induce them to express their newly acquired but previously dormant committed state. Thus, initiation and promotion parallel events during normal development and differentiation, but during carcinogenesis the new cell populations are aberrant in terms of specialized functions and growth control.

Changes in epidermal growth factor receptors associated with adenovirus transformation, chemical carcinogen transformation and exposure to a phorbol ester tumor promoter

Abstract Transformation of rat embryo cells by wild type or a mutant (H5ts125) of human adenovirus type 5 results in >97% reduction in binding of epidermal growth factor to cell surface receptors. A 26 to 89% reduction in binding was observed in clones of NIH-3T3 cells transformed by benzo(a)pyrene (BP) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Chemically or spontaneously transformed rat liver epithelial cell lines and a rat hepatoma cell line also displayed low binding. The reduction of epidermal growth factor binding does not correlate quantitatively with expression of other markers of transformation. Brief exposure to a phorbol ester tumor promoter reduced epidermal growth factor binding in all of these cell types.

Reaction Mechanisms and Chemical Networks — Types of Elementary Steps and Generation of Laminar Mechanisms*

A theory was introduced recently [O. Sinanoglu, J. Am. Chem. Soc. 97 (1975) 2309] which systematizes chemical kinetics giving relations between numbers of intermediates, catalysts, reactants and products, and numbers of elementary reaction steps in any and all possible mechanisms. In fact, there are finite numbers of and manageably few mechanisms a priori possible in any chemical or biochemical system for finite numbers ofsteps or species. The present paper gives the details of the method for obtaining all a priori mechanisms in which each intermediate species occurs in at most two of the elementary steps. These mechanisms are called laminar while all others, including autocatalytic ones are turbulent (treated in another paper). A structural induction method given allows one to deduce the possible intermediates as actual chemicals, from the networks. Both laminar and turbulent types of elementary steps possible are listed and discussed. A priori possible laminar mechanisms and possible observed overall reaction types, including the

Transition-probability theory of cell proliferation and a biochemical approach to the kinematics of the cell cycle

Abstract Cell proliferation as a result of a chain of biochemical events in the cell is manifested biologically in four cytologically discernible proliferating phases ( G 1 , S , G 2 , M ) of the cell cycle, a nonproliferating phase ( G 0 ) and a D phase of cells in either dying or differentiating stages. In terms of transitional biochemical events in the cell, patterns of cell loss and variations of cell duration are related to the basic mechanisms of cell proliferation. Based on the stochastic transition of cell numbers in the subphases of the cell cycle, a probability distribution is found to be asymptotically a time-dependent multinomial distribution. The dynamic equations of cell proliferation in terms of their transitions between phases are derived from the expectation values of this probability distribution. Correlations of cell subphases are found to be constant, while variances are functions of time. Mitosis labeling and the cell growth curve are predicted with a suitable set of biochemical parameters. Thus, this approach can be used as a starting point for correlating biochemical mechanisms of cell proliferation with cell kinetics, which is basic to cancer therapy and an understanding of the nature of neoplastic growth.

Canonical formulation of the biochemical cell cycle and its statistical prediction of cellular parameters

Abstract Cell proliferation is analyzed by means of the transition probability between biochemical events in the subphases of the cell cycle. The initial distribution of the cell population, whether synchronized or random, can influence the early proliferation rate of the cells. The generalized growth rate is a linear combination of many modes of cell cycle which are defined by an eigenvalue system. With the inclusion of nonproliferating phases, the stationary state, where the total number of proliferating cells remains constant but the total cell number increases, is solved for under a constraint on the transition matrix. The steady state where all subphase populations grow in a definite ratio is largely determined by the eigenvector of the highest eigenvalue of the transition matrix. A statistical estimation of biological parameters associated with the cell cycle, such as DNA content, cell size, or total proteins, is made using a combination of the eigenvectors of the transition matrix and the distribution of the observable. This analysis considers the effects of nonproliferating phases in the estimation of biological parameters and various experimental conditions. A canonical formulation is established for nonconservative open systems which have nonhermitian transition matrices and nonconservative cell numbers.