Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.

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In this paper we describe the synthesis and characterization of a series of simple spermine/amino acid conjugates, some of which potently inhibit the uptake of spermidine into MDA-MB-231 breast cancer cells. The presence of an amide in the functionalized polyamine appeared to add to the affinity for the polyamine transporter. The extensive biological characterization of an especially potent analogue from this series, the Lys-Spm conjugate (31), showed this molecule will be an extremely useful tool for use in polyamine research. It was shown that the use of 31 in combination with DFMO led to a cytostatic growth inhibition of a variety of cancer cells, even when used in the presence of an extracellular source of transportable spermidine. It was furthermore shown that this combination effectively reduced the cellular levels of putrescine and spermidine while not affecting the levels of spermine. These facts together with the nontoxic nature of 31 make it a novel lead for further anticancer development.

Background: Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual 11 translational frameshift mechanism that responds to polyamine levels.

The prostate gland has among the highest level of polyamines in the body and prostate carcinomas have even greater elevated polyamine levels. These ubiquitous molecules synthesized by prostate epithelium are involved in many biochemical processes including cellular proliferation, cell cycle regulation, and protein synthesis. These properties have made polyamines a potential target for therapeutic intervention in diseases of excessive cell proliferation such as cancer. However, attempts to limit tumor growth by inhibition of polyamine synthesis have not been very successful since cells have the capacity to take up polyamines from the bloodstream. We report here studies utilizing polyamine depletion by means of a combination of blockade of polyamine synthesis with DFMO (a-di¯uoromethylornithine), an inhibitor of ornithine decarboxylase, the rate limiting enzyme in the polyamine synthetic pathway, and ORI 1202, a novel inhibitor of polyamine transport into the cell. This cytostatic combination, even in the presence of excess extracellular polyamines, signi®cantly slowed the growth of the human tumor cell line PC-3 grown in tissue culture with an EC 50 in the mM range. Other prostate cell lines were similarly growth inhibited including LNCaP.FGC and DU145. Growth of the PC-3 tumor cell line as a xenograft in nude mice was also slowed signi®cantly by this combination of compounds. Polyamine levels in the tumor were lowered from control tumor levels. This combination therapy could provide an effective and potentially non-toxic therapy for prostate tumors. Prostate Cancer and Prostatic Diseases (2000) 3, 275±279.