Paul O. P. Ts’o

Nonionic Oligonucleotide Analogs as New Tools for Studies on the Structure and Function of Nucleic Acids Inside Living Cells

Two types of nonionic oligonucleotide analogs, deoxyribonucleotide alkyl phosphotriesters and deoxyribooligonucleoside methylphosphonates, have been synthesized to serve as selective inhibitors of cellular nucleic acid function. The backbones of these analogs are resistant to nuclease hydrolysis and the analogs are taken up by mammalian cells and certain bacterial cells in culture. Sequence specific analogs inhibit tRNA aminoacylation and translation of mRNA in both mammalian and bacterial cell-free systems in a specific manner as a result of oligomer binding to complementary sequences of the target nucleic acid. These analogs also inhibit cellular protein synthesis and growth of living cells. Selective inhibition of bacterial versus mammalian cell growth is observed with a methylphosphonate oligomer complementary to the Shine-Dalgarno sequence of 16S rRNA. Methylphosphonate complementary to the 5’-end of U1RNA and to the donor splice site of SV40 large T antigen pre-mRNA inhibit T-antigen production in SV40-infected cells.

Control of Gene Expression by Oligonucleoside Methylphosphonates

A major goal in understanding the processes of aging, cancer and differentiation is to understand gene expression and thus the function of various proteins in the overall biochemical processes of the cell. One of the classical ways to study gene expression is through the use of temperature sensitive mutants. Although this approach has been particularly effective in studying gene expression in bacteria and viruses, it is technically more difficult in eukaryotes, particularly mammalian cells. It would be desirable to have an alternative approach which would allow selective inhibition of gene expression either at the level of transcription or at the mRNA level. Recent studies have shown that such regulation may be achieved through the use of complementary DNAs (cDNAs) or anti-sense RNAs. The expression of mRNA can be regulated both in the test tube and in cells by cDNAs which selectively hybridize to a target mRNA. Control of cell-free mRNA translation in this manner is termed hybridization arrest (1). This procedure has been used to study the location and arrangement of adenovirus 2 genes within the viral genome and to analyze mRNA populations in mouse liver (2). Hybridization arrest has also been used to study the function of the 3′-non-coding region of globin mRNA (3).

Progress in the Study of in Vitro Neoplastic Transformation

Since the pioneering work of Berwald and Sachs1,2 in 1963–1965, much effort has been made to develop adequate and useful mammalian cell systems for the study of the basic mechanisms of neoplastic transformation in chemical carcinogenesis. This constitutes a major aspect of this chapter. More recently, advances have been made to characterize neoplastic transformation at the molecular level; i.e. the study on neoplastic transformation has advanced from the animal model to the cell system, and then to the molecular description. While this approach is still in its infancy, some promising results have begun to appear. The description of this future trend will be included at the end of this chapter.

Neoplastic Transformation, Somatic Mutation, and Differentiation

One of the most dreaded diseases of our time is cancer, the topic of this symposium. This disease originates from a single cell, or groups of cells, which become neoplastic or tumorigenic. Also, as indicated from the topics of this symposium, this neoplastic transformation often has a large input from external/environmental factors, particularly man-made. This dread disease then becomes closely related to our personal and economic lives and has a large impact on health care and the economic activities of our society.

Involvement of Radicals in Chemical Carcinogenesis

Chemical carcinogens comprise a large and structurally diverse group of synthetic and naturally occurring compounds. It appears almost axiomatic that such compounds must react with tissue components in order to induce neoplastic transformation. With the exception of the carcinogenic alkylating agents, most chemical carcinogens are not reactive per se and must be converted to reactive forms either chemically or metabolically. Data are emerging to indicate that electrophilic reactants are the ultimate form of most, if not all, chemical carcinogens (Miller, 1970). The proximate forms of a number of chemical carcinogens might be convertible to free radicals, i.e., electrophilic reactants, and suggest a possible role for the free radical in carcinogenesis. The presence of free radicals in tobacco smoke has been demonstrated (Lyons and Spence, 1960; Bluhm et al., 1971) and the increased incidence of lung cancer in cigarette smokers based on epidemiological studies is well known. The chemical and metabolic conversion of a number of chemical carcinogens to free radicals and the interaction of these radicals with DNA will be described in this communication.

On the Development of New Chemotherapeutic Agents in the Coming Decade

Division of Biophysics The Johns Hopkins University Baltimore, Maryland The major advances in scientific research which currently have a significant impact on medicine can be generally described in two areas: 1) application of instrumentation such as laser beam surgery and NMR imaging, etc. and 2) the understanding of biology and medicine at the molecular and atomic levels. For the development of new therapeutic agents, the second area certainly is the focal point of attention. Thus, the major impact of the molecular sciences on the medical and pharmaceutical industries is the topic of this short essay.

Understanding Cancer — the Need for a Broad and Integrated Scientific Approach

Understanding and controlling cancer is a challenge to science equal to the challenge of controlling hereditary disorders and understanding the aging process. Several aspects of the disease are considered in making such a comprehensive statement.

Cellular Studies on the Interrelationship Among Cancer, Aging and Cellular Differentiation

An experimental animal model and cellular system, based on the Syrian hamster (Mesocricetus auratus), has been developed for the study of the interrelationship among cancer, aging and differentiation. Using this system, we have identified a progenitor-like cell which is present in primary and low passage mesenchymal cell cultures. The loss or absence of these cell.s is correlated with reduced in vitro proliferative capacity (cellular senescence), reduced response to growth promoting factors, and reduced susceptibility to in vitro neoplastic transformation. Based on these studies we propose a hypothesis on the central role of stem cells and/or progenitor cells in the interrelationship among cancer, aging and cellular differentiation.

The Use of Cell Cultures to Assay the Effects of Chemicals on Bone Marrow

Fresh bone marrow and liquid cultures of marrow were exposed to MNNG, 4NQ0, MAMA, or PDD. MNNG was toxic in all systems; 4NQ0 was either toxic or stimulatory depending upon concentration and type of cell system; MAMA and PDD stimulated cell proliferation. Differentiation was directed to either myeloid or myelomonocytic pathways.

Nuclear Magnetic Resonance Studies of Nucleic Acids and Proteins

With the advances in spectrometer instrumentation and computer technology, NMR has become a very powerful technique in the study of biopolymers, such as nucleic acids and proteins. This approach can potentially provide information about the properties and interactions of biopolymers at the atomic level through investigation of the magnetic properties of many nuclei, particularly those having spins of 1/2, such as 1H, 13C, 19F, and 31P. To most effectively utilize this approach, however, we must define (1) the type of information which can be gained using NMR, (2) the type of questions to be posed about the characteristics of proteins and nucleic acids at the atomic level, and finally (3) the kinds of improvements needed in NMR studies for the future. The biochemist is mostly concerned not only with the nuclei which were mentioned previously, but also with 11B, 15N, and 14N.