Michael I. Sherman

Differentiation-defective mutants of mouse embryonal carcinoma cells: Response to hexamethylenebisacetamide and retinoic acid

We have generated by mutagenesis eight differentiation-defective sublines from three murine embryonal carcinoma (EC) cell lines. These mutants grossly resemble parental cells in the absence of inducers of differentiation. Based upon response to retinoic acid (RA) or hexamethylenebisacetamide (HMBA), the mutants can be grouped into three types: (a) RA-selected cells that lack cellular RA binding protein (cRABP) activity and fail to differentiate in response to RA or HMBA; (b) RA- or HMBA-selected cells that possess cRABP but differentiate poorly, if at all, in the presence of RA or HMBA; and (c) cells originally selected for lack of response to HMBA but which retain cRABP and the ability to differentiate in response to RA.

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Abstract Incorporation of [ 35 S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N -sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N -sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.

Antisense and antiviral therapy.

HIV is a complex virus and AIDS is a difficult disease to contend with. In view of this, the speed with which basic research and clinical discoveries have contributed to our understanding of HIV and AIDS has been impressive. In considerable part, this is because AIDS has become prevalent in the midst of the biotechnology explosion. On the other hand, current therapeutic strategies for combatting AIDS are still relatively unsophisticated. It will be interesting, in retrospect, to examine how ultimate therapeutic approaches to this disease have been influenced by opportunities that our new-found technology base allows. One of the innovative approaches being considered as an anti-AIDS therapeutic strategy is the use of antisense oligonucleotides. The concept of using antisense sequences to block gene expression dates back several years and has been used against numerous genetic targets (see ref. 1). Indeed, in 1986, Zamecnik et aI.* first reported the feasibility of the approach for HIV, at least for infected cells in culture. As the other articles in Part I11 of this volume attest, we are becoming progressively more sophisticated in our ideas of how to exploit the antisense strategy effectively for therapeutic purposes. One can now imagine the use of DNA antisense oligonucleotides to block expression of viral mRNA or more complex antisense structures and strategies that would interfere with either retroviral replication or transcription of provirus. Conjugated oligonucleotides have been designed that variously lead to enhanced binding to, covalent binding to, or degradation of, the target nucleic acid sequence (reviewed by Cohen and Zon’). Combined ribozyme-antisense technology is another example of a more elaborate approach that has already been shown to elicit cleavage of HIV viral RNA in a catalytic manner.3 The antisense strategy is particularly suited to application in anti-HIV and other antiviral therapies because it should be possible to target unique viral sequences that do not have functional counterparts in the human genome. In theory, then, an antisense therapy could have exquisite specificity. As is so often the case with new therapeutic approaches, there are significant difficulties that must be overcome before antisense therapy can become a reality. Although the strategy might be novel, the major problems are not fundamentally different from generic ones that must be addressed in the development of any therapeutic, namely how to generate an active, safe, and cost-effective drug. A number of technical issues are relevant to such concerns, including optimization of potency of antisense molecules, determination of the most effective and safest ways of getting antisense sequences into cells, evaluation of the pharmacokinetics of antisense mole-

Effects of Retinoids on Growth and Differentiation

The retinoids constitute a group of compounds which includes vitamin A (retinol), its metabolites and a multitude of synthetic analogues. Retinol can be oxidized to retinal and these two retinoids are essential for vision and fertility. Apart from eliciting these activities upon cells of the visual and reproductive systems, retinoids appear to have a much more general influence upon cellular behavior: they affect the propensity to differentiate and proliferate. Much of the information to support this view is derived from studies with cultured cells (reviewed in 1). However, it was recognized long ago (2,3) that in animals on diets lacking retinoids, epithelial cells showed evidence of both metaplasia and hyperplasia.