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Six benzoylphenylurea (BPU) derivatives have been synthesized in Japan and extensively evaluated by the U.S. National Cancer Institute. They demonstrated potent antitumor activity in vitro against several cancer cell lines as well as in vivo against several tumor models. One of these agents, NSC639829, has now entered clinical trials. Studies have shown that these compounds are effective inhibitors of in vitro tubulin polymerization. The parent compound, NSC624548 (HO-221), has been shown to inhibit calf thymus DNA polymerase alpha activity. In this study we examined the effects of four BPU derivatives (NSC624548, NSC639828, NSC639829, and NSC654259) on the activity of the synthesome-associated DNA polymerase alpha, Escherichia coli DNA polymerase I, and calf thymus DNA polymerase alpha. Among the compounds tested, only NSC624548 and NSC639828 inhibited the activities of E. coli DNA polymerase I and calf thymus DNA polymerase alpha. Excess DNA polymerase I or DNA polymerase alpha dramatically reduced the inhibition produced by these compounds. NSC624548 and NSC639828 also showed inhibitory effects of the synthesome-associated DNA polymerase alpha similar to that produced upon using the purified E. coli and calf thymus enzymes. All of the four compounds did not show inhibitory effect on DNA polymerase delta. The similar pattern of inhibition these compounds exert on both the purified calf thymus and the synthesome-associated DNA polymerase alpha offers further support for the validity of the DNA synthesome as a novel in vitro model system for studying anticancer drug action.

A considerable effort is being made to discover more effective anticancer agents and to develop rapid, accurate, and reliable methods for evaluating the usefulness of the new compounds and analyzing their mechanisms of action. In this regard, our laboratory was the first to report the isolation, characterization, and extensive purification of an intact, stable, and fully functional mammalian cell multiprotein complex, which we designated the DNA synthesome. The DNA synthesome has been isolated from a wide variety of mammalian cells as well as from tissues and has been shown to be fully competent to support SV40 origin-specific and large T antigen-dependent DNA replication in vitro. In this article we will review the utilization of the DNA synthesome as a powerful in vitro model system for analyzing the mechanism of action of S-phase specific anticancer agents.

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468., Methods: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta)., Results: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug., Conclusions: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.

Purpose: Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis., Methods: We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects ofdFdCTP and araCTP on the synthesome-associated DNA polymerase alpha and delta activities., Results: Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 microM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full-length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 microM dCTP, 3 microM dFdCTP and 60 microM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases alpha and delta. It was found that the drug concentrations required to inhibit 50% of the synthesome-associated DNA polymerase delta activity were much higher than those required to inhibit 50% of DNA polymerase alpha activity for both dFdCTP and araCTP., Conclusion: Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase alpha activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.

A model of chronic entrapment neuropathy in the rabbit sciatic nerve was developed to try to elucidate the pathogenesis of chronic nerve entrapment. A non-compressive Silastic cuff was wrapped around the nerve at the mid-thigh level in eight rabbits. A sham operation that only elevated the sciatic nerve was performed in seven control rabbits. Six months later, the blood flow in the cuffed and the control sciatic nerves was determined with intra-arterially injected microspheres. Blood flow was significantly reduced in the entrapped nerve, compared to control nerves, but only in the segment proximal to the cuff. After surgical release of the cuff, the blood flow significantly increased in the proximal segment. This suggested that decreased blood flow may occur, but not necessarily at the site of nerve entrapment. Nerve conduction velocity changes were also consistent with an entrapment neuropathy. However, there were no significant quantitative morphometric changes at the nerve entrapment site, that is, in myelin thickness/nerve diameter ratio, distribution of axon sizes, and mean nerve fiber diameter. This indicates that only a mild entrapment was created.

Twenty-seven cases of replantation for avulsion injuries of the thumb from 1979 to 1987 were analyzed retrospectively. Inclusion criteria required all thumbs to have tendinous attachments (either the extensor pollicis longus or the flexor pollicis longus) to the amputated part. Thirteen replants survived for an overall survival rate of 48%. Avulsions at or proximal to the metacarpophalangeal joint had a survival rate of 83% compared with a 38% survival rate distal to the metacarpophalangeal joint. This difference was not statistically significant. Of the 13 patients with surviving replanted thumbs, 11 (85%) achieved protective sensation only and 2 attained a two-point discrimination <10 mm. Of the 13 replanted thumbs that survived, 7 were treated with primary arthrodesis, 4 at the level of the interphalangeal joint and 3 at the metacarpophalangeal joint. The other 6 thumbs were treated with primary or secondary tendon transfers. In a literature review of thumb avulsion injuries, survival rates range from 26% to 100%. In this study, tendon transfers and primary arthrodesis were useful adjuncts in the management of thumb avulsions, and the majority of patients with successfully replanted thumbs attained protective sensation only. Larger studies are needed to better define statistically significant relationships affecting replantation survival.

The majority of human intestinal intraepithelial lymphocytes (HIELS) express CD8+, and the T cell Receptor (TCR) alpha beta. A minority of HIELS utilize TCR gamma delta chains. V delta 1 is established as the TCR-delta expressed by most TCR gamma delta HIELS. Since V delta 1 is the dominant intestinal TCR and V gamma (I) family is preferentially used in forming a heterodimer, this study was conducted to characterize individual V gamma (I) utilization in HIELS. Intestinal lymphocytes were isolated from four samples of colonic epithelium obtained from patients undergoing colon resection or endoscopy. RNA was isolated and cDNA synthesized. PCR amplification was performed with consensus J gamma and V gamma primers in these regions. PCR products were cloned and sequenced. All samples had V gamma 4 transcripts, a majority V gamma 3 whereas V gamma 2 and V gamma 8 were less frequent. No V gamma 2 transcripts had any predicted TCR protein products. Similarly, very few potentially productive V gamma 3 transcripts were found. In contrast, almost all V gamma 4 transcripts were found to be in-frame and the only V gamma 8 transcript was in-frame. The CDR3 region of the gamma transcripts were small compared to published intestinal TCR delta recombinations. All CDR3 regions contained at least one charged amino acid. The limited number of functional transcripts adds evidence to the oligoclonality of intestinal TCRs expressing the TCR V gamma (I) family. The short CDR3 regions support the concept of limited antigen recognition by this lymphocyte population.

Intestinal cell water was studied in rats that have been fasted then refed, two conditions that are known to decrease and increase, respectively, proliferation of the intestinal epithelium. Cell water decreased (15%) during fasting and returned to normal with refeeding. The amiloride-sensitive sodium uptake, which estimates uptake through the Na+/H+ exchange was higher in the ileum than the jejunum, but the jejunal uptake, unlike the ileal, was significantly increased in fasted/refed rats than in normally fed or fasted ones. Osmotic shrinkage of intestinal cells followed by restitution of their cell volume stimulated the Na+/H+ exchange in all of the three groups of animals, but the increase was most prominent in the fasted and the fasted/refed groups. Also, shrinkage of cultured jejunal crypt cells (IEC-6) by a hypertonic solution increased intracellular alkalinization that was inhibited by amiloride. The results provide evidence for a relationship between the change in intestinal cell size, such as that which occurs during fasting/refeeding, and the activation of the Na+/H+ antiport system. This may represent one of the signals that initiates intestinal proliferation in the fasting/refeeding state.

Our technique combines the advantages of two proven techniques of the wraparound flap and vascularized joint transfer while offering a more normal thumb, both functionally and cosmetically. Its advantages are as follows: 1. A more normal-looking thumb with good length 2. Preservation of motion through joint transfer 3. Maintenance of growth potential through transfer of vascularized epiphyses 4. Minimal donor-site morbidity