Jack Cohen

GENES AND DEVELOPMENT

This chapter describes genes and their development. Development of nearly all animals is determined up to the phyletic stage, by the spatial organization of the egg cytoplasm and its mRNA, proteins, and other important molecules that were stockpiled during oogenesis. These early embryological processes are usually fuelled by food and energy reserves that have also been passed from the mothers surplus. Liver, kidney, or skin cells all have nuclei with the same genes; yet they all exhibit different forms and functions, in terms not only of the major proteins secreted or contained but also in their shapes, sizes, division rates, susceptibility to radiation, or cell poisons. Theories of differentiation used to start by assuming that cells were initially similar and attempt to account for later differences. The acquisition of differentiation may be sudden, but is usually progressive. The other case in which de-differentiation has been postulated is that of regeneration. Because all individuals of a species have different cell antigens the virus cannot mutate to mimic the natural antigens, because these vary from individual to individual as do the antibodies which recognize them.

Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV)

Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.

EMBRYOS AND EVOLUTION

This chapter describes embryos and their evolution. Animal evolution has produced and is producing a great variety of animal forms. The ways in which this variation appears in the life history of the animals concerned is part of the province of embryology. Caenogenetic modifications are those variations that benefit the young form, embryo or larva, and have no particular significance for the adult. Very often they are lost at hatching, birth, or metamorphosis. The converse situation, paedomorphosis, is the early acquisition of the adult state while the organism still has the larval modifications and general body form. The insects are probably derived by paedomorphosis from a form resembling modern millipedes, which have a six-legged stage in their embryology. The vertebrates may have been derived from echinoderm-like ancestors by retention of their larval characters. In many ways the primates, and especially the anthropoid apes and man, may be said to be neotenous mammals. Deviation, on the other hand, describes those changes that are adaptations, primarily by adults, to meet adult requirements. Because of the conservative nature of developmental changes, the early stages of two related animals usually resemble one another more than later stages.

Oligodeoxynucleotide analogs with 5'-linked anthraquinone

We report here the synthesis of novel 5′‐linked oligodeoxynucleotides, both normal phosphodiester and phosphorothioate analogs, in which a covalently attached group at the 5′‐terminus is an anthraquinone. These compounds represent a new class of antisense compounds in which the base sequence of the oligodeoxynucleotide serves to deliver a nuclease‐resistant reactive drug‐like molecule to a cellular target nucleic acid (mRNA or DNA).

Studies on the mechanism of selective retention of porphyrins and metalloporphyrins by cancer cells.

Comparative studies of the toxicity, stability, and retention of the water-soluble porphyrin, tetraphenylporphyrin sulfonate (TPPS), and its complex with Mn(III), have been made with the human breast cancer cell line MCF-7 wild type, and an adriamycin-resistant line derived from it, termed AdrR. Based on growth inhibition, we determined the maximum non-toxic concentration of MnTPPS tolerated by these cells. The integrity of MnTPPS in vitro was investigated by fluorescence microscopy, and we found that there is very little dissociation of MnTPPS within these cells within 4 days. We report novel proton magnetic resonance relaxation measurements of the bulk water of cells in a gel matrix undergoing perfusion. A slightly greater net uptake of MnTPPS in the wild-type cells was observed compared to AdrR; however, there was no significant difference in retention of MnTPPS. These results indicate that over a period of several hours the mechanism of selective retention of these compounds in tumour cells is not due to specific interaction with heme-binding protein, of which there is enhanced expression in the resistant cells. The fact that the net rate of washout of MnTPPS is approximately the same as the net rate of uptake also appears to eliminate compartmentalization or enzymatic modification of MnTPPS within these cells.

Metabolic studies of mammalian cells by 31P-NMR using a continuous perfusion technique

Levels of ATP and Pi in metabolically active Chinese hamster lung fibroblasts were monitored noninvasively by 31P-NMR over many hours and under a variety of conditions. The cells were embedded in a matrix of agarose gel in the form of fine threads which were continuously perfused in a standard NMR tube. The small diameter of the thread allows rapid diffusion of metabolites and drugs into the cells. The changes in ATP and Pi levels were followed as a function of time in response to perfusion with a glucose-containing medium, with isotonic saline and with a medium containing 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. This gel-thread perfusion method should enable routine NMR studies of cellular metabolism, and may have other potential biological applications.

Concerning antisense inhibition of the multiple drug resistance gene.

Recently, Vasanthakumar and Ahmed reported (Vasanthakumar, G.; Ahmed, N.K., Cancer Communications 1:225-232; 1989) a complete inhibition of the multiple drug resistance gene (MDR1) in the K562/III erythroleukemia cells, using a 15 bases-long methylphosphonate oligodeoxynucleotide analog. The sequence used, however, contained three mismatches relative to the corresponding fragment of the human MDR1 gene and, hence, the results reported cannot at present be regarded as a classical antisense effect. We have made attempts to inhibit the expression of the MDR1 gene in MCF-7 human breast cancer cells selected for resistance to Adriamycin using phosphorothioate analogs of oligodeoxynucleotides. Studies with model 35S-labeled-phosphorothioates indicated poor uptake of the compounds into the cells; the radioactivity was located mainly in the soluble fraction (cytoplasm), but membranes and the nuclear fraction were also labeled. Unmodified oligodeoxynucleotides were toxic to the cells, whereas the phosphorothioates were not. The MDR1 inhibition with phosphorothioates was studied by measuring their effects on adriamycin toxicity and by immunocytochemical titration of P170. Elevation of adriamycin cytotoxicity consistent with a decreased drug resistance was observed with one antisense sequence, but the immunocytochemical assay indicated only slight inhibition of the synthesis of P170. In the wild type (drug sensitive) MCF-7 cells phosphorothioates decreased adriamycin toxicity in a sequence-independent manner. The results indicate that the effects of antisense oligodeoxynucleotides on cells are complex. Computer simulation of the secondary structure of MDR1 mRNA indicated not only extensive folding but, also, the presence of many regions not involved in intramolecular hybridization, which are of potential interest as targets for antisense oligodeoxynucleotides.

Monitoring the transport and phosphorylation of 2‐deoxy‐D‐glucose in tumor cells in vivo and in vitro by 13C nuclear magnetic resonance spectroscopy

We describe the use of 2‐deoxy‐D‐[6‐13C]glucose to follow simultaneously, by 13C NMR, both transport and phosporylation to its 6‐phosphate form, in MCF‐7 breast cancer cells in vitro and in vivo in subcutaneous tumors in nude mice.

Versatile interactive graphics display system for molecular modelling by computer.

THE intricacies of constructing molecular models of macro-molecules based on coordinates determined by X-ray crytallography has become a major task in itself, particularly now that a large number of protein structures has been determined. The combination of a computer and a graphics display terminal can be a powerful tool in studies of macro-molecules. Several computer-based systems for the display of molecular structures have been described which differ greatly1–5 and it was recently suggested that smaller computers can provide adequate capability6.

Monitoring intracellular metabolism by nuclear magnetic resonance

Publisher Summary This chapter discusses the monitoring intracellular metabolism by nuclear magnetic resonance. MRS (magnetic resonance spectroscopy) and positron emission tomography (PET) scanning are the only two methods employed in radiology that are basically metabolic in their mode of operation. This chapter discusses various methods for NMR studies of intracellular metabolism. 31 p and 13 C NMR spectroscopy have been used to monitor bioenergetics and phospholipid metabolism of several human tumor cell lines, both embedded in gel threads and subcutaneously implanted in nude mice. It is important to note that the 31 p spectra obtained for the same cell line in both of these two environments— namely, perfused cells embedded in basement membrane gel thread and tumor in a nude mouse are very similar. This to some extent justifies the use of cell perfusion to study intracellular metabolism as being relevant to the in vivo situation. The applications that follow will focus on the studies of bioenergetics and phospholipid metabolism.