Dynamics of recurrent viral infection.

In chronic viral infection, low levels of viral replication and infectious particle production are maintained over long periods, punctuated by brief bursts of high viral production and release. We apply well-established principles of modelling virus dynamics to the study of chronic viral infection, demonstrating that a model which incorporates the distinct contributions of cytotoxic T lymphocytes (CTLs) and antibodies exhibits long periods of quiescence followed by brief bursts of viral production. This suggests that for recurrent viral infections, no special mechanism or exogenous trigger is necessary to provoke an episode of reactivation; rather, the system may naturally cycle through recurrent episodes at intervals which can be many years long. We also find that exogenous factors which cause small fluctuations in the natural course of the infection can trigger a recurrent episode. Our model predicts that longer periods between recurrences are associated with more severe viral episodes. Four factors move the system towards less frequent, more severe episodes: decreased viral infectivity, decreased CTL efficacy, decreased memory T cell response and increased antibody efficacy.

The interferon-inducible 204 gene is transcriptionally activated by mouse cytomegalovirus and is required for its replication.

Infection of cells with viable or UV-inactivated murine cytomegalovirus (MCMV) increased the IFN-inducible 204 gene at both the mRNA and the protein levels. The activity of a reporter gene driven by the mouse Ifi204 promoter induced following virus infection showed that this increase was due to transcriptional activation. Moreover, FACS analysis of infected mouse embryo fibroblasts (MEF) stably transfected with a p204-dominant-negative mutant (p204dmMEF) revealed that they do not accumulate at the G1/S border in the same way as infected MEF transfected with the empty vector (neoMEF). MCMV DNA synthesis is significantly delayed (144 h in p204dmMEF vs 72 h in neoMEF), due to retarded expression of viral genes, namely, IE1 and DNA polymerase, as shown by Western blot comparison of p204dmMEF and neoMEF extracts. These results demonstrate that MCMV may exploit the Ifi204 gene to regulate the cell cycle and enhance its DNA synthesis.

The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation.

We have previously demonstrated that overexpression of p204, a member of the Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibroblasts (MEF) inhibits cell proliferation, but does not affect cell growth in MEF derived from Rb-/- mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo fibroblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a significant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this effect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-alpha antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb-/- cells were unsensitive to the IFN-alpha induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-alpha antiproliferative activity and (ii) the primary target of p204 leading to efficient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system.

Analysis of messages expressed by Echinostoma paraensei miracidia and sporocysts, obtained by random EST sequencing.

A lambdaZAP Express cDNA library was constructed with mRNA obtained from immature miracidia within eggs, hatched miracidia, and sporocysts of Echinostoma paraensei. This cDNA library was amplified and 213 expressed sequence tag (EST) sequences (averaging 466 nucleotides in length) were obtained. The mean percentage of unresolved bases within the EST sequences was 0.4%, ranging from 0 to 4.6%. The 213 ESTs represent 151 unique messages. BLAST (version 2.0.8) analysis disclosed that 64 unique E. paraensei messages (42.4%) had significant similarities (BLAST score < or =e-5), at deduced amino acid or nucleotide levels, with known sequences in the nonredundant GenBank databases or the dbEST database (NCBI). The remainder, 57.6% of the unique EST-encoded messages, scored nonsignificant hits. Most of the E. paraensei messages that could be assigned a cellular role based on sequence similarities were involved in gene/protein expression. Several ESTs scored highest similarities with sequences obtained from trematode species. A total of 22,560 nucleotides present in open reading frames from ESTs that aligned with known sequences was used to determine codon usage for E. paraensei. Analysis of a subset of eight ESTs that contained full-length open reading frames did not reveal a bias in codon usage. Also, EST sequences were found to contain 3' untranslated regions with an average length of 69.9 +/- 88.4 nucleotides (n = 46). The EST sequences were submitted to GenBank/dbEST, adding to the 51 available Echinostoma-derived sequences, to provide reference information for both phylogenetic analysis and study of general trematode biology.

Evidence from two planorbid snails of a complex and dedicated response to digenean (echinostome) infection.

The planorbid snail Biomphalaria glabrata responded to exposure to either the compatible digenetic trematode Echinostoma paraensei or the incompatible species Echinostoma trivolvis by producing increased amounts of several distinctive plasma polypeptides. These polypeptides characteristically precipitated from plasma when mixed with secreted-excreted products (SEP) of sporocysts or rediae from either digenean species. In contrast, control snails, or snails that had been wounded or infected with bacteria (Serratia marcesens or Staphylococcus epidermidis) showed no obvious plasma alterations and no precipitates formed when their plasma was mixed with SEP. Another planorbid species, Helisoma trivolvis, which displays reverse compatibility for the echinostome species used, also responded to exposure to both echinostomes by increased production of plasma polypeptides that precipitated in the presence of SEP. With some individual variation, these 2 snail species synthesized SEP-reactive plasma polypeptides forming diffuse bands centred at 53, 65, 80-120 and 200 kDa (the latter absent in Helisoma trivolvis). The 53 kDa polypeptides had not been observed before, whereas the others have been noted from B. glabrata. The diffuse 65 kDa band was strongly bound by anti-fibrinogen antibodies, supportive of earlier studies indicating it contains fibrinogen-related domains. The other specified polypeptides were also bound by these antibodies raising the possibility that they too contain fibrinogen domains. The results are suggestive of a general ability of these 2 planorbid snails to detect the presence of echinostomes even if the latter are subsequently incapable of development. The complex response they then mount, one not evoked by other challenges such as wounding or bacterial infection, may represent a dedicated response to a frequently encountered group of pathogenic parasites, the digeneans (echinostomes).

Overexpression of cellular dihydrofolate reductase abolishes the anticytomegaloviral activity of methotrexate.

Cytomegalovirus (CMV) stimulates numerous cellular pathways upon infection. One of these pathways involves activation of dihydrofolate reductase (DHFR), an essential enzyme in the biosynthesis of purines and thymidylate. Here we report that methotrexate (MTX), an inhibitor of DHFR, suppresses murine CMV replication at the level of DNA synthesis in quiescent NIH 3T3 cells. However, MTX has no antiviral activity in NIH 3T3 sublines resistant to MTX due to DHFR overexpression. These results directly link MTX antiviral activity to DHFR and demonstrate that DHFR plays an essential role for CMV replication in quiescent cells.

The interferon-inducible 204 gene, a member of the Ifi 200 family, is not involved in the antiviral state induction by IFN-alpha, but is required by the mouse cytomegalovirus for its replication.

To examine whether Ifi 200 genes are involved in antiviral state induction by IFNs we expressed mutant forms capable of inactivating the endogenous p204 and analyzed replication of both RNA and DNA viruses following IFN-alpha treatment. Inactivation of p204 does not impair replication of vesicular stomatitis virus, encephalomyocarditis virus, ectromelia virus, and herpes simplex virus 1 and does not alter an IFN-alpha induced antiviral state. By contrast, in cells lacking functional p204, mouse cytomegalovirus (MCMV) replication is strongly inhibited and is not further modulated by IFN-alpha. These results suggest that p204, a member of the Ifi 200 gene family, is not involved in the IFN-alpha-induced antiviral activity against some RNA or DNA viruses, but is required by MCMV for its replication.

Demonstration of multiple HPV types in laryngeal premalignant lesions using polymerase chain reaction and immunohistochemistry.

Recent evidence has shown that human papillomavirus (HPV) is involved in both the development of carcinoma and in premalignant mucosal lesions of the oral cavity. This study examined the relationship of HPV infection to some pathological features in precancerous lesions of the larynx, not examined extensively so far. Fifty formalin-fixed paraffin-embedded tissue sections containing human laryngeal precancerous lesions were screened for the presence of HPV infection by polymerase chain reaction, and for capsid protein expression by immunohistochemistry with polyclonal antibody directed against the L1 protein. The presence of HPV DNA was detected in 28 of 50 specimens (56%), including 9/12 cases with mild dysplasia (75%), 3/6 cases with moderate dysplasia (50%), and 7/11 cases with severe dysplasia (64%). Multiple HPV infections, containing two or three types, were detected in 17 of the 28 HPV-positive lesions (60%). Of 21 cases with keratosis and no dysplasia, 11 were positive for HPV DNA (52%) and 4 showed L1 staining (36%). By contrast, L1 positivity was revealed only in two lesions with moderate dysplasia, confirming that fully productive HPV infection is strictly dependent on epithelial differentiation and surface keratinization. The probability that HPV is a cofactor in the malignant progression of these lesions is suggested by the fact that 3/4 patients who developed cancer within 50 months were positive for HPV DNA.

The HMG protein T160 colocalizes with DNA replication foci and is down-regulated during cell differentiation.

The high mobility group protein T160, the murine homolog of the human structure-specific recognition protein 1, was first supposed to be involved in the process of V-(D)-J recombination, since it could bind to recombination signal sequence probes. We have recently cloned T160 by using an unrelated DNA probe and shown that it binds to either cruciform or linear DNA with no sequence specificity. In this work, we performed a detailed analysis of T160 expression and immunolocalization. We show that T160 is a phosphoprotein broadly conserved from yeast to mammals, with a high level of expression in all the cell lines tested and in tissues containing a high degree of proliferating cells. Indirect immunofluorescence analysis by confocal laser microscopy revealed that T160 distribution in the cell nucleus is not uniform, and focus-like staining was observed. Cell cycle studies by BrdU incorporation suggest that the appearance of T160 nuclear foci is specific of mid to late S phase. Furthermore, while T160 expression does not change during the cell cycle, it is dramatically down-regulated when cells begin to differentiate, as highlighted in C2C12 myoblasts and myotubes. The disappearance of T160 nuclear staining in multinucleated myotubes is shown. Taken together, these data suggest that its function may be less specific than V-(D)-J recombination and more related to some cellular basic process, such as DNA replication or repair.

Human cytomegalovirus stimulates cellular dihydrofolate reductase activity in quiescent cells.

Human cytomegalovirus (HCMV) productively infects quiescent fibroblasts in which the levels of deoxynucleotide triphosphates (dNTPs) and cell functions involved in DNA metabolism are very low. Since sufficient dNTPs levels are essential for human HCMV replication, host cell enzymes involved in the biosynthesis of dNTPs might be expected to be stimulated by viral infection in quiescent cells. We report that HCMV infection of quiescent fibroblasts stimulates the activity of cellular dihydrofolate reductase (DHFR), a key enzyme in DNA precursor synthesis. We also demonstrate that suppression of DHFR activity by the specific inhibitor methotrexate prevents HCMV replication and DNA synthesis. These observations indicate that induction of DHFR activity by HCMV is required for efficient viral replication in quiescent fibroblasts.

In vitro and in vivo expression analysis of the interferon-inducible 203 gene.

The interferon (IFN)-inducible protein family 200 is encoded by structurally related genes located on mouse chromosome 1. The encoded proteins so far characterized and designated p202, p204, and pD3 contain at least one copy of a conserved 200 amino acid domain in addition to other regions that are different or missing among the various family members. We have recently characterized a cDNA clone (203 cDNA) encoding a 408 amino acid protein bearing structural similarities to p202 and p204. Here, we report its pattern of expression in vitro and in vivo. In vitro, the mRNA and protein encoded by the 203 gene were increased by IFN-alpha in several cell lines of different histologic origin. By contrast, no significant induction was observed in vivo in mice from C57BL/6 and BALB/c strains even after treatment with the IFN-inducer poly rI:rC. In addition, the constitutive expression of 203 gene was restricted to some myeloid and lymphoid tissues, namely, thymus, bone marrow, and spleen. Comparison of the expression pattern of the 203 and 202 genes in three mouse strains revealed that they exhibit a differential inducibility by IFN and a reciprocal expression pattern. The 203 mRNA was constitutively expressed in C57BL/6 and BALB/c mice and undetectable in the spleen of DBA/2 mice. The 202 mRNA was strongly induced by poly rI:rC in the spleen of DBA/2 and BALB/c mice but absent in C57BL/6 mice. Southern analysis revealed a restriction fragment length polymorphism in the 203 locus. Taken as a whole, these results demonstrate a remarkable difference in the in vivo IFN responsiveness of two members belonging to the same gene family with a similar degree of IFN inducibility in vitro. Moreover, the reciprocal expression pattern in C57BL/6 and DBA/2 mice could mean that p203 and p202 play the same role in a mouse strain in which only one of them is expressed.

Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.

The high-mobility group protein T160 binds to both linear and cruciform DNA and mediates DNA bending as determined by ring closure.

The high-mobility group protein T160 was isolated by screening a phage library from a murine pre-B-cell line L1210. South-Western experiments have previously shown that this protein binds to V-(D)-J recombination signal sequences, suggesting that it may be a sequence-specific DNA-binding protein. However, neither gel-shift nor footprinting analyses have been successfully employed with the T160 protein, despite an extensive effort. In this study, the T160 protein or truncated forms made soluble through denaturing and renaturing cycles in urea were successfully used in gel-shift experiments showing that T160 binds to cruci-form or linear duplex DNA with no apparent sequence specificity. Furthermore, fragments longer than 100 bp efficiently formed covalently closed circular monomers in the presence of T160 and T4 DNA ligase, indicating that the protein is capable of introducing bends into the duplex. Last, tissue distribution by Western blotting analysis showed that the T160 protein is expressed in various murine tissues in addition to those of lymphoid origin. Considering its broad evolutionary conservation (from plants to mammals) also, these results suggest that the functional role of the T160 protein is not limited to V-(D)-J recombination, but might be involved in basic processes such as DNA replication and repairing, where irregular DNA structures are generated and very likely recognized by HMG domain proteins.

A family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection.

After infection with the digenetic trematode Echinostoma paraensei, hemolymph of the snail Biomphalaria glabrata contains lectins comprised of 65-kDa subunits that precipitate polypeptides secreted by E. paraensei intramolluscan larvae. Comparable activity is lacking in hemolymph of uninfected snails. Three different cDNAs with sequence similarities to peptides derived from the 65-kDa lectins were obtained and unexpectedly found to encode fibrinogen-related proteins (FREPs). These FREPs also contained regions with sequence similarity to Ig superfamily members. B. glabrata has at least five FREP genes, three of which are expressed at increased levels after infection. Elucidation of components of the defense system of B. glabrata is relevant because this snail is an intermediate host for Schistosoma mansoni, the most widely distributed causative agent of human schistosomiasis. These results are novel in suggesting a role for invertebrate FREPs in recognition of parasite-derived molecules and also provide a model for investigating the diversity of molecules functioning in nonself-recognition in an invertebrate.

Suppression of high mobility group protein T160 expression impairs mouse cytomegalovirus replication.

The high mobility group (HMG-1) box proteins bind both non-B-DNA conformations and specific nucleotide sequences. They have been implicated in a wide variety of cellular functions involving DNA, such as transcription, replication and recombination. To determine whether HMG-1 box protein T160 plays a role in virus replication, we employed an antisense strategy to inhibit its expression in NIH 3T3 cells. The two T160 clones that expressed levels of T160 50% lower than those expressed by clones transfected with the empty vector (Neo+ clones) were investigated with respect to their permissiveness to the growth of viruses representing three families: Rhabdoviridae, vesicular stomatitis virus (VSV); Picornaviridae, encephalomyocarditis virus (EMCV), and Alpha- and Betaherpesviridae, herpes simplex virus 1 (HSV-1) and mouse cytomegalovirus (MCMV), respectively. They displayed a high degree of resistance to MCMV replication, but were fully permissive to the other viruses. Competitive PCR and probing IE-1 products by Western blot analysis showed that this resistance was not due to depressed levels of virus adsorption during the early phases of infection. We therefore conclude that T160 is involved in replication of the betaherpesvirus MCMV.

Decreased expression of the high-mobility group protein T160 by antisense RNA impairs the growth of mouse fibroblasts.

The T160 protein belongs to the HMG-1 box protein family and preferentially binds to non-B-DNA conformations with no sequence specificity. Its exact role has yet to be defined, though it seems to participate in processes involving DNA, such as replication, transcription and recombination. We have used an antisense RNA strategy to investigate its role in cell growth and proliferation. T160 expression is strongly suppressed by stable introduction of an antisense construct into NIH3T3 cells, and this decrease is accompanied by substantial changes in the growth properties of the stable transfectants. Impaired growth of T160- cells was mainly related to two mechanisms: i) decreased rates of cell proliferation at normal serum concentration; and ii) occurrence of cell death by apoptosis at low serum concentration, as demonstrated by both flow cytometry and microscopy. The finding that decreased T160 availability affects cell proliferation, provides further evidence of its involvement in a basic cell function, such as DNA replication.

Excision of 2',2'-difluorodeoxycytidine (gemcitabine) monophosphate residues from DNA.

The activity of gemcitabine (dFdC), an effective agent against solid tumors, depends on the incorporation of its triphosphate into DNA. In vitro investigations demonstrated that, depending on the sequence of template DNA, polymerases may pause after incorporation of gemcitabine nucleotide at either the 3'-terminal or 3'-penultimate position. Proofreading enzymes such as 3'-->5' exonucleases, which are associated with DNA polymerases, can excise mismatched deoxynucleotides from DNA. To model this reaction, we evaluated excision of the gemcitabine nucleotide from oligodeoxynucleotide (19-mer) containing 3'-penultimate dFdC monophosphate (dFdCMP) or dCMP by the 3'-->5' exonuclease of the Klenow fragment. The rate of excision of the 3'-terminal deoxynucleotide was similar, with both primers resulting in formation of primers with terminal dCMP or dFdCMP. The primer containing dCMP was further excised, and by 40 min, more than 75% of total radioactivity was in excision products smaller than 18-mer. In contrast, most of the primers (90%) with terminal dFdCMP were unexcised. When primers terminated with either dFdCMP or dCMP were used as substrates, normal primer was hydrolyzed almost completely by 20 min; however, only 40% of primers containing dFdCMP had excision of dFdCMP molecule. Kinetic studies demonstrated that the enzyme had similar affinity for primers containing penultimate or terminal dFdCMP, but the apparent Vmax for excision was 4-5-fold greater for removal of a 3'-terminal deoxynucleotide than for cleavage of a dFdCMP molecule. Reaction conditions that permitted polymerization of one deoxynucleotide to primers containing either 3'-penultimate dCMP or dFdCMP were used to evaluate excision during DNA synthesis. The excised primers could not be extended because the reaction lacked the requisite deoxynucleotide triphosphate. After 5 min, more than one-half of the dCMP primers were extended, whereas only 15% had been excised. In comparison, 30% of the analogue-containing primers lost the terminal deoxynucleotide, with a proportional lower incidence of extension (30%). Lesser excision of dFdCMP-containing substrate was observed in reactions containing deoxynucleotide triphosphates required to make full-length products. Consistent with this result, in the absence of 3'-->5' exonuclease activity, both primers were extended similarly by the polymerization unit of the Klenow fragment. Taken together, these data demonstrate that dFdCMP residues are difficult to excise from DNA, and DNA polymerase can extend primers with 3'-dFdCMP. This results in the internal incorporation of dFdCMP into DNA, as observed in whole cells.

[Comprehensive life style changes by coronary patients--an intervention study]. / Umfassende Lebensstiländerung bei Koronarkranken--eine Interventionsstudie.

In an intervention trial 31 coronary patients participated in a comprehensive lifestyle change program and 43 control patients received the usual care of the conventional cardiac rehabilitation system. First preliminary findings show that the patients in the intervention group not only improved their health related lifestyle but also increased their exercise capacity and reduced depression, while there was no substantial improvement in the control group. Up to the present, the existing data indicate that German heart patients are able to make comprehensive lifestyle changes and that these changes have positive effects on biomedical and psychosocial variables.

Ab initio quantum mechanical and X-ray crystallographic studies of gemcitabine and 2'-deoxy cytosine.

Gemcitabine 2',2'-difluoro 2'-deoxy cytosine (GEM) is a novel nucleoside which has demonstrated broad preclinical anti-cancer activity and appears promising in early stage human clinical trials. One purpose of this study was to characterize the energetically favored conformational modes of GEM by means of ab initio quantum mechanical studies with comparison to a novel X-ray crystallographic structure, and to determine the performance of ab initio quantum mechanical theory by comparison with X-ray structural data for GEM and 2'-deoxy cytosine (CYT). Another objective of this study was to attempt to determine key structural and electronic atomic interactions relating to the 2',2'-difluoro substitution in GEM by the application of ab initio quantum mechanical methods. To our knowledge, these are the first reported ab initio quantum mechanical geometry optimizations of nucleosides using large (e.g. 6-31G*) slit valence function basis sets. The development of accurate physicochemical models on a small scale enables us to extend our studies of GEM to more complex studies including DNA incorporation, deamination, ribonucleotide reductase inhibition, and triphosphorylation.

Radiosensitization of pancreatic cancer cells by 2',2'-difluoro-2'-deoxycytidine.

PURPOSE: We have reported that the deoxycytidine analog 2',2'difluoro-2'-deoxycytidine (dFdCyd) is a potent radiosensitizer of HT29 human colon cancer cells probably through its effects on intracellular deoxyribonucleotide (dNTP) pools. Because dFdCyd has activity against pancreatic cancer in clinical trials, we wished to determine if dFdCyd would radiosensitize human pancreatic cancer cells. METHODS AND MATERIALS: We assessed the effect of dFdCyd on radiation sensitivity of two human pancreatic cancer cell lines, Panc-1 and BxPC-3. To begin to investigate the mechanism of sensitization, we determined the effect of dFdCyd on dNTP pools and cell cycle distribution. RESULTS: We found that dFdCyd produced radiation enhancement ratios of 1.7-1.8 under noncytotoxic conditions in both cell lines. Sensitization was not associated with intracellular levels of 2',2'-difluoro-2'-deoxycytidine triphosphate, the cytotoxic metabolite of dFdCyd, but occurred when dATP pools were depleted below the level of approximately 1 micromolar. Although both cell lines showed substantial cell cycle redistribution after drug treatment, the flow cytogram of the BxPC-3 cells would not, by itself, be anticipated to result in increased radiation sensitivity. CONCLUSIONS: These findings demonstrate that dFdCyd is a potent radiation sensitizer of human pancreatic cancer cells and support the development of a clinical protocol using combined dFdCyd and radiation therapy in the treatment of pancreatic cancer.