Opposing effects of cyclosporin A and tyrphostin AG-1478 indicate a role for Src protein in the cellular control of mineralization

Cyclosporin A (CsA) induces osteoporosis but not through direct activation of osteoclasts. CsA also inhibits cell-mediated mineralization in marrow stromal cell culture, whereas the tyrphostin AG-1478 increases mineralization. These antagonistic effects on mineralization were used to discern molecules that underwent phosphorylation changes in association with their opposing effects on mineralization. In parallel, quantitative changes in Src protein were followed. Multiple dexamethasone (DEX)-stimulated stromal cell cultures were grown with and without a mineralization-inhibiting dose (0.1 μM) of CsA and were harvested on different days of DEX stimulation. Immunoblots of gel-fractionated cell extracts showed that the most noticeable changes in tyrosine phosphorylated proteins (TPP) were seen on day 8 of DEX stimulation. At least 15 TPP bands, mostly smaller than 53 kDa, were more prominent in CsA-treated cultures on day 8. Under CsA, Src protein quantity decreased on day 8, but its cleavage product (52/54 kDa) was sixfold more abundant then on day 7. Day 8 was chosen to test the effect of AG-1478 on the CsA-induced TPP changes. Dimethyl sulfoxide (DMSO) alone, the solvent of AG-1478, increased mineralization in CsA-treated versus CsA-untreated cultures and slightly decreased Src and its cleavage product. AG-1478 at 5 μM, in CsA cultures increased the specific alkaline phosphatase activity threefold, with a slight change in mineralization relative to controls grown with DMSO alone. This was accompanied by decreased intensity of several TPP bands smaller than 36 kDa. In contrast, treatment with 50 μM of AG-1478 increased the intensity of TPP bands at the same molecular size range. This high AG-1478 dose decreased cell counts selecting mineralizing cells. The results indicate that increased Src protein cleavage product on day 8 by CsA is associated with mineralization inhibition, which is opposed by DMSO and 50-μM AG-1478, thus antagonizing the effect of CsA on mineralization. Direct or indirect interaction between Src and TPP, antagonistically affected by CsA and AG-1478, is likely to underlay cellular control of mineralization. Changes in p19 and p29 intensity showed association with mineralization that was reflected by a significant direct and inverse correlation, respectively, with calcium precipitation per cell. J. Cell. Biochem. 71:116–126, 1998. © 1998 Wiley-Liss, Inc.     

淫羊藿苷促进体外培养骨髓间充质细胞成骨性分化活性强于蛇床子素

比较研究蛇床子素与淫羊藿苷处理对体外培养的大鼠骨髓间充质细胞(rat bone marrow stromal cell, rBMSC)成骨性分化的影响.从体外分离培养的大鼠骨髓间充质细胞,筛选出最佳的蛇床子素和淫羊藿苷处理的浓度为1×10-5 mol/L, 然后用最佳的浓度对体外培养的大鼠骨髓间充质细胞进行药物干预;在药物干预后的第3、6、9、12和15 d后测定碱性磷酸酶活性（alkaline phosphatase,ALP）和钙含量;第12 d 进行钙化结节茜素红染色;第12 h、24 h、48 h、72 h和96 h 对OXS、Runx-2、骨形态发生蛋白(bone morphogenetic protein,BMP-2)和collagen-I mRNA 表达水平进行real-time RT-PCR检测.结果显示,浓度为1×10-5 mol/L蛇床子素和淫羊藿苷干预均可提高体外培养的骨髓间充质细胞ALP活性,增加Ca含量,提高Runx、OXS、BMP-2和collagen-1 mRNA的表达水平.同时,淫羊藿苷在促进体外培养骨髓间充质细胞成骨性分化活性强于蛇床子素.     

Studies of the Levamisole inhibitory effect on rat stromal-cell commitment to mineralization

The ability of Levamisole to decrease mineralization in skeletal tissue is usually related to its effect on alkaline phosphatase (ALP). However, Levamisole is also suspected to diminish mineralization by an additional mechanism which is unrelated to the ALP control of apatite crystal growth. To delineate the time in differentiation during which Levamisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells were propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0.2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. This suggested the existence of a Levamisole-sensitive mechanism for mineralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Levamisole decreased Day 21 mineralization when added on Days 0, 3, 5, and 7, but not when added on Day 9. The Levamisole-induced inhibition of mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to mineralize is determined during the first week of culture. The early inhibitory effect of Levamisole on mineralization was associated with increased Day 12 ALP activity.     

Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.     

Analysis of cell-mediated mineralization in culture of bone-derived embryonic cells with neurofibromatosis

von Recklinghausen neurofibromatosis (NF1) is an autosomal dominant genetic disorder associated with congenital pseudoarthrosis and with short stature. To examine whether the NF1 phenotype includes functional osteogenic defects, embryonic bone-derived cells affected with NF1 were tested in culture for specific alkaline phosphatase (ALP) activity and cell-mediated mineralization and compared with other embryonic bone derived cells. NF1 showed a relatively higher specific ALP activity, which has further increased in response to dexamethasone + β-glycerophosphate (βGP) (Dex medium) coordinately with a decrease in cell proliferation. In the control group, two samples showed increased ALP activity, one showed decreased activity and the forth one did not show any change in ALP. NF1 cells were distinguished from other cells regarding day 21 mineralization, they did not mineralize when cultured with ascorbate alone in the absence of Dex medium, whereas control cells did mineralize. Adding βGP resulted in mineralization by NF1 cells but less than in other cells. In addition, NF1 cells responded to dexamethasone by increasing the βGP-induced mineralization, as opposed to cells from other embryonic bones, which either did not respond or have even decreased mineralization under dexamethasone. Upon cis-hydroxyproline exposure, Dex medium has also distinguished NF1 cell ALP activity from that of other cell origins. Inhibition of respiratory complex II by malonate showed that most embryonic bone-derived cells of 12 weeks gestation are malonate resistant; thus, malonate selection was ineffective. This is in contrast to rat marrow stromal cells previously shown to undergo mineralizing cell enrichment in response to malonate. Exposure to levamisole, of Dex-treated cells, at days 0–11 has inhibited day 21 mineralization in all tested cultures in spite of the increase in day 11-specific ALP activity. Both malonate and levamisole did not distinguish NF1 cells from the osteogenic phenotype of other cells. Essentially embryonic bone-derived cells from 12 weeks gestation, cultured in the absence of βGP, retained their mineralization capacity, which does not increase under dexamethasone, as distinguished from NF1 cells which require βGP for mineralization and positively respond to dexamethasone. Therefore, bone-derived NF1 cells may be useful for studying the regulation of the mineralization process.     

Characteristics of heterologous antisera against stromal fibroblasts]

As shown with the use of heterologous rabbit antifibroblast sera (AFS) to stromal mechanocytes of guinea pig, the media from the cultured bone marrow, spleen, and thymus stromal fibroblasts contained specific trypsinoresistant fibroblast protein (AG-1), which was also present in the normal blood serum and on the surface of stromal fibroblasts. AG-1 was insensitive to collagenase effect and apparently differed from the chief surface protein of fibroblasts (CSP). AG-1 is referred to gamma1-globulin, and is probably a new specific surface fibroblast protein. AFS contained also antibodies to the nonspecific protein of fibroblasts (AG-2), alpha1,-globulin related to AG1.     

Increased radiation-induced apoptosis of Saos2 cells via inhibition of NFkappaB: a role for c-Jun N-terminal kinase

To elucidate the possible effect of NFkappaB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFkappaB constitutive inhibitor, mIkappaB (Saos2-mIkappaB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mIkappaB cells but not in Saos2-EV control cells, with intact NFkappaB activity. We find as expected, that this NFkappaB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFkappaB signaling in Saos2-mIkappaB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFkappaB resulted in decreased expression of the DNA damage protein GADD45beta, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mIkappaB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-c from mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mIkappaB cells. Antisense inhibition of GADD45beta in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFkappaB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma.     

The effect of tyrphostins AG494 and AG1478 on the autocrine growth regulation of A549 and DU145 cells

We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFR's inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true.     

EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway

The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by approximately 50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser(235/236). Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.     

Differential induction of cell-mediated mineralization in rat marrow stroma by sera from women of low and high risk for vertebral fracture

The purpose of this study was to analyze the ability of sera to reflect the state of bone metabolism by testing the osteogenic response of mesenchymal cells in culture. Sera of 20 peri- and postmenopausal women were tested before the initiation of hormone replacement therapy. The responding cells were osteoprogenitors (OPC) of rat marrow stroma which normally respond to dexamethasone (DEX) and β-glycerophosphate (βGP) by proliferation, differentiation, and mineralization in culture. Instead of DEX, diluted sera (1:50) were applied to rat stromal cell cultures for analysis of their ability to affect cell proliferation, specific alkaline phosphatase (ALP) activity, and cell-mediated mineralization. The results were compared individually with the respective values of vertebral bone mineral density (BMD), expressed as the number of standard deviations above or below the mean BMD of reference populations (positive or negative Z-score). Serum donors were divided in two; the group with positive Z-scores was considered to have a low risk, and that with negative Z-scores was considered to have a higher risk for vertebral fractures. No significant difference was found between the two groups in the ability of their sera to induce cell proliferation or specific ALP activity. However, sera representing negative Z-scores induced sixteenfold less mineralization than those of positive Z-scores. The scatter of individual mineralization values was highly discriminatory between the two groups (α < 0.00). These results indicate that the serum-induced, cell-mediated mineralization in culture might be suitable for initial evaluation of fracture risk and thus deserve further investigation. © 1996 Wiley-Liss, Inc.     

Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells

The nonadherent (NA) population of bone-marrow-derived mononuclear cells (MNC) has been demonstrated to be a source of osteogenic precursors in addition to the plastic-adherent mesenchymal stromal cells (MSC). In the current study, two subpopulations of late adherent (LA) osteoprogenitors were obtained by subsequent replating of NA cells, and their phenotypic, functional, and molecular properties were compared with those of early adherent (EA) MSC. Approximately 35% of MNC were LA cells, and they acquired a homogeneous expression of MSC antigens later than EA cells. In EA-MSC, the alkaline phosphatase (ALP) activity increased significantly from time of seeding to the first confluence, whereas in LA cells it raised later, after the addition of mineralization medium. All subpopulations were able to produce type I collagen and to deposit extracellular matrix with organized collagen fibrils. The proportion of large colonies with more than 50% of ALP positive cells as well as the calcium content was higher in LA than in EA cells. Molecular analysis highlighted the upregulation of bone-related genes in LA-MSC, especially after the addition of mineralization medium. Our results confirm that bone marrow contains LA osteoprogenitors which exhibit a delay in the differentiation process, despite an osteogenic potential similar to or better than EA-MSC. LA cells represent a reservoir of osteoprogenitors to be recruited to gain an adequate bone tissue repair and regeneration when a depletion of the most differentiated component occurs. Bone tissue engineering and cell therapy strategies could take advantage of LA cells, since an adequate amount of osteogenic MSCs may be obtained while avoiding bone marrow manipulation and cell culture expansion.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

Lumican regulates osteosarcoma cell adhesion by modulating TGFβ2 activity

Human osteosarcoma cell lines were recently shown to express and secrete the small leucine rich proteoglycan (SLRP) lumican, with the ability to regulate the growth and motility of these cells. In this study, lumican-deficient Saos 2 cells were demonstrated to have increased adhesive capability onto fibronectin (FN) (p≤0.01). Upon neutralization of endogenous transforming growth factor β2 (TGF-β2) activity, no difference in the ability of lumican siRNA-transfected and scramble siRNA-transfected Saos 2 cells to adhere onto FN was detected (p=NS). Exogenous TGF-β2 was shown to stimulate Saos 2 cell adhesion to FN (p≤0.01). These results therefore, suggest that the inverse correlation existing between lumican expression and Saos 2 cell adhesion is dependent on active TGF-β2 signaling. Furthermore, the significant increase in Smad 2 activation present in lumican-deficient cells (p≤0.01) was annulled in the presence of the anti-TGF-β2 peptide, demonstrating that lumican is an upstream regulator of the TGF-β2/Smad 2 signaling cascade. Crucial to FN-dependent adhesion, β1 integrin expression and pFAK activation were likewise identified as downstream TGF-β2 effectors regulated by lumican expression. In conclusion, this study demonstrates a novel out-in signaling circuit in human osteosarcoma cells: secreted to extracellular matrix lumican is an endogenous inhibitor of TGF-β2 activity, resulting in downstream effector modulation including pSmad 2, integrin β1 and pFAK to regulate osteosarcoma adhesion.     

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at 10(-8) M and maximal at 10(-7) M). Northern blot analysis showed that the statin (10(-7) M) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (10(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Lumican expression is positively correlated with the differentiation and negatively with the growth of human osteosarcoma cells

Osteosarcoma is the most common primary bone tumour associated with childhood and adolescence. The possible role of the small leucine-rich proteoglycan, lumican, in the growth and metastasis of various cancer types has recently been investigated. In this study, the expression of lumican was examined in moderately differentiated (MG-63) and well-differentiated (Saos 2) human osteosarcoma cell lines of high and low metastatic capability, respectively. Real-time PCR, western blotting with antibodies against the protein core and keratan sulfate, and specific enzymatic digestions were the methods employed. The two human osteosarcoma cell lines were found to express and secrete lumican partly substituted with keratan sulfate glycosaminoglycans. Importantly, the non-metastatic, well-differentiated Saos 2 cells produced lumican at rates that were up to sevenfold higher than those of highly metastatic MG-63 cells. The utilization of short interfering RNA specific for the lumican gene resulted in efficient down-regulation of its mRNA levels in both cell lines. The growth of Saos 2 cells was inhibited by lumican, whereas their migration and chemotactic response to fibronectin were found to be promoted. Lumican expression was negatively correlated with the basal level of Smad 2 activation in these cells, suggesting that lumican may affect the bioavailability of Smad 2 activators. By contrast, these cellular functions of highly aggressive MG-63 cells were demonstrated not to be sensitive to a decrease in their low endogenous lumican levels. These results suggest that lumican expression may be positively correlated with the differentiation and negatively correlated with the progression of osteosarcoma.     

Effect of arabinofuranosylthymine on the replication of Epstein-Barr virus and relationship with a new induced thymidine kinase activity

1-beta-D-Arabinofuranosylthymine (araT) is a selective inhibitor of Epstein-Barr virus replication induced in both thymidine kinase (TK)-negative (TK-) and TK+ variants of the lymphoid cell line P3HR-I. This analog has no effect on the growth of noninduced cells (T. Ooka and A. Calender, Virology 104:219-223, 1980). The synthesis of early antigens is not affected by the analog, whereas that of late viral capsid antigens is completely inhibited, as demonstrated by the indirect immunofluorescence technique; kinetic reassociation experiments have also shown that araT strongly inhibits replication of viral DNA. Phosphorylation of the tritiated form of the analog ([3H]araT) was analyzed by thin-layer chromatography in cultures of control and induced cells, and the results demonstrated that only induced cells can convert the analog to the triphosphate form. These results indicate that the selective effect of araT in induced cells is probably related to a new virally induced TK activity. Preliminary characterization of this new activity has shown that it is able to phosphorylate the analog specifically, whereas cellular TKs cannot. araTTP, a final phosphorylation product of araT, is a potent inhibitor of Epstein-Barr virus-specific DNA polymerase, suggesting a possible inhibitory action of this product on Epstein-Barr virus replication.