Stimulation of cartilage zones of the calf costochondral growth plate in vitro by growth hormone dependent rat plasma somatomedin activity

The actions of rat plasma somatomedin activity dependent on growth hormone were investigated in vitro on separated zones of cartilage from the calf costochondral junction. Plasma somatomedin maximally stimulated the uptake of[3H]thymidine into cartilage cells of the proliferating region. Cartilage deeper in the growth plate possessed the highest uptake of [35S]sulphate which was also stimulated by somatomedin. Somatomedin, therefore, appears to promote both cell replication and matrix synthesis throughout the growth plate cartilage although the two processes were greatest in different cartilage regions. Growth hormone or tri-iodothyronine did not directly alter the uptake of either isotope into the growth plate cartilage.     

Response of chondrocytes isolated from human foetal cartilage to plasma somatomedin activity

Plasma somatomedin activity enhanced the incorporation of [3H]thymidine into chondrocytes isolated from human foetal cartilage during weeks 13-21 of gestation. Human growth hormone (0.1-20 muu./ml), human placental lactogen (0.1-5 microgram/ml) and insulin (0.25-10 muu./ml) had no direct effects on the synthesis of DNA in these chondrocytes, although insulin at concentrations of 2.5-100 mu./ml increased [3H]thymidine incorporation by up to 400%.     

Increased thymidine incorporation into fetal rat cartilage in vitro in the presence of human somatomedin, epidermal growth factor and other growth factors

The incorporation of [3H]thymidine by rat costal cartilage in vitro was studied at different fetal and postnatal ages and the effect of partially purified human somatomedin, mouse epidermal growth factor, platelet secretion products, insulin and growth hormone on thymidine uptake by fetal cartilage was examined. Thymidine uptake in plasma-free medium was many times greater in late fetal life than after birth. The incorporation of [3H]thymidine into costal cartilage from 21-day fetuses was significantly (P less than 0.05) increased above control values in the presence of 10 micrograms somatomedin/1, and when cartilage was incubated in medium containing somatomedin and diluted human plasma there was a synergistic action. Epidermal growth factor at a concentration of 1 ng/l was a potent stimulator of thymidine uptake. Secretion products from human platelets after their aggregation by thrombin stimulated [3H]thymidine uptake at a concentration of 2% (v/v), but were inhibitory at high concentrations. High concentrations of platelet secretion products stimulated the incorporation of [35S]sulphate by cartilage. A pharmacological concentration of 10 mu. insulin/ml stimulated [3H]thymidine uptake, but not concentrations of 1 or 100 mu./ml. Growth hormone had no effect. The results showed that fetal cartilage had a greater endogenous mitogenic activity than postnatal cartilage. While somatomedins may be important in the regulation of fetal body growth, other protein growth factors also stimulate fetal skeletal tissues.     

低硒、低碘及联合对亲代和子代大鼠骨、软骨生长的影响

目的 研究低硒、低碘及其联合对亲代和子代大鼠骨、软骨生长的影响.方法 48只断乳SD健康大鼠,按体质量随机分对照组、低硒组、低碘组和低硒低碘组,每组12只.4组大鼠分别采用人工配制的低硒、低碘、低硒低碘及含硒、碘量正常的饲料喂饲.大鼠饲养8周后(约3月龄)进行组内繁殖;亲代大鼠喂饲至6月龄、子代大鼠喂饲至3月龄时,取亲代和子代大鼠血样,测定血清硒及T3、T4;取大鼠右侧胫骨及左侧膝关节.用游标卡尺测定胫骨长度、中段额状横径、胫骨上端关节软骨横径、纵径;膝关节包埋切片后,光镜下测定胫骨近端生长板厚度、生长板软骨肥大层、增殖层细胞层数.结果 低硒对亲代和子代大鼠血清硒有明显影响(F值分别239.56、232.68,P均<0.05),对于代血清T4水平也有明显影响(F=6.95,P<0.05);低碘对亲代和子代大鼠血清T3、T4均有明显影响(F值分别为14.11、14.05,30.29、34.53,P<0.01),低硒、低碘联合对子代大鼠血清T4水平的影响存在交互作用(F=5.99,P<0.01).亲代和子代大鼠血清硒,低硒组[(30.28±6.34)、(43.95±9.75)μg/L]、低硒低碘组[(30.33±5.18)、(35.40±3.06)μg/L]均明显低于低碘组[(345.83±29.55)、(245.24±9.95)μg/L]、对照组[(358.64±30.50)、(236.50±9.75),P均<0.05].低硒低碘组[(0.55±0.05)、(0.88±0.14)mmol/L]亲代和子代大鼠血清T3均明显低于对照组[(0.75±0.08)、(1.26±0.26)mmol/L,P均<0.05],低碘组[(24.11±2.29)、(42.10±8.92)mmol/L]、低硒低碘组[(20.66±1.93)、(26.55±5.98)mmol/L]亲代和子代大鼠血清T4均明显低于对照组[(36.15±2.74)、(52.79±8.84)mmol/L]和低硒组[(28.12±3.33)、(52.02±11.99)mmol/L,P均<0.05];低硒低碘组子代大鼠血清T4明显低于低碘组(P<0.05).子代大鼠中,低硒,低碘对胫骨长度、生长板厚度、增殖细胞层数、肥大细胞层数影响明显(F值分别为24.31、6.98,40.76、56.15,25.24、82.82,10.07、5.57,P<0.01或<0.05).低硒和低碘对胫骨长度、生长板厚度、增殖细胞层数、肥大细胞层数的影响存在交互作用(F值分别为5.68、24.86、41.82、9.12,P<0.05或<0.01);低硒组[(33.17±0.34)mm]、低硒低碘组胫骨长度[(31.30±0.87)mm]明显低于对照组[(34.12±0.32)turn,P均<0.05];低硒低碘组生长板厚度[(1.60±0.18)mm、增殖细胞层数(8.54±0.81)、肥大细胞层数(4.95±0.37)明显低于低硒组[(3.03±0.10)mm、14.68±0.84、6.60±0.31]、低碘组[(2.90±0.09)mm,13.75±0.33、6.61±0.84]及对照组[(3.19±0.09)mm、14.94±0.36、6.64±0.26,P均<0.05)];低碘组生长板厚度、增殖细胞层数小于对照组(P<0.05).结论 低硒影响子代大鼠胫骨生长,低碘影响子代大鼠软骨细胞增殖,降低生长板厚度,低硒低碘联合显著影响子代大鼠骨、软骨的生长.     

Platelet-derived growth factor and multiplication-stimulating activity II, but not multiplication-stimulating activity III-2, stimulate [3H]thymidine and [35S]sulphate incorporation by fetal rat costal cartilage in vitro

The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0.21-21 micrograms/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10-100 micrograms/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5-10 micrograms/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides.     

Characterization of a specific somatomedin-c receptor on isolated bovine growth plate chondrocytes

The interaction of somatomedin (Sm) with growth plate chondrocytes (GPCs) is believed to be the primary stimulus of skeletal growth. Using techniques designed to disrupt as little as possible the phenotypic characteristics of GPCs, we have been able to obtain 3-4 x 10(8) viable cells from the major physes of one newborn calf. The availability of these cells plus essentially pure Sm-C/insulin-like growth factor I, the most GH-dependent Sm, has now made possible detailed studies of the interaction of this radiolabeled peptide with the GPC receptor and of the subsequent processing of this hormone by these cells. The enzymatic methods required to free GPCs from their matrix led to loss of receptors, followed by rapid receptor regeneration by de novo synthesis in suspension cultures. Binding of [125I]iodo-Sm-C to GPCs was time dependent and saturable, with optima at 15 C and pH 7.8. At 37 C, binding peaked at 90 min and declined thereafter. Multiplication-stimulating activity, insulin, and nerve growth factor were less potent than unlabeled Sm-C in competition with [125I]iodo-Sm-C for its receptor. Human GH, epidermal growth factor, and fibroblast growth factor failed to show competition even at 10(-6) M. Analysis of the fate of [125I]iodo-Sm-C bound to GPCs at 37 C provided evidence that this hormone is internalized and extruded from the cell in a partially degraded form. Scatchard analysis of [125I]iodo-Sm-C binding to GPCs and to chondrocytes isolated from articular cartilage revealed similar Ka values, but reproducibly 2-6 times more receptors on growth plate than on articular chondrocytes.     

The role of the resting zone in growth plate chondrogenesis

In mammals, growth of long bones occurs at the growth plate, a cartilage structure that contains three principal layers: the resting, proliferative, and hypertrophic zones. The function of the resting zone is not well understood. We removed the proliferative and hypertrophic zones from the rabbit distal ulnar growth plate in vivo, leaving only the resting zone. Within 1 wk, a complete proliferative and hypertrophic zone often regenerated. Next, we manipulated growth plates in vivo to place resting zone cartilage ectopically alongside the proliferative columns. Ectopic resting zone cartilage induced a 90-degree shift in the orientation of nearby proliferative zone chondrocytes and seemed to inhibit their hypertrophic differentiation. Our findings suggest that resting zone cartilage makes important contributions to endochondral bone formation at the growth plate: 1) it contains stem-like cells that give rise to clones of proliferative chondrocytes; 2) it produces a growth plate-orienting factor, a morphogen, that directs the alignment of the proliferative clones into columns parallel to the long axis of the bone; and 3) it may also produce a morphogen that inhibits terminal differentiation of nearby proliferative zone chondrocytes and thus may be partially responsible for the organization of the growth plate into distinct zones of proliferation and hypertrophy.     

The ontogeny of growth hormone receptors in the rabbit tibia

To address the question of the mode of action of GH in stimulating longitudinal bone growth, we have used a panel of anti-GH receptor monoclonal antibodies to demonstrate GH receptors in the rabbit tibia and have studied the ontogeny of these receptors. In the neonate, receptors were localized in the hypertrophic zone between the cartilage canals, a region that develops into a secondary ossification center. In support of this finding, receptors were also localized on monolayer cultures of human infant costal chondrocytes. In 20- and 50-day-old rabbits, receptors were localized on reserve and proliferative chondrocytes in the growth plate. In 50- and 130-day-old rabbits receptors were localized on proliferative chondrocytes in the condylar cartilage. In older (180-day-old) rabbits with closed growth plates, GH receptors could not be detected, even in condylar cartilage. These results support the case for revision of the somatomedin hypothesis to accommodate a direct interaction between GH and receptors on epiphyseal chondrocytes.     

Effect of growth hormone and insulin-like growth factor I (IGF-I) on the expression of IGF-I messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo

Conflicting data exist as to whether insulin-like growth factor I (IGF-I) messenger RNA (mRNA) and peptide are expressed within chondrocytes. This question is pertinent to the mode of GH action on longitudinal bone growth. We have, therefore, investigated this issue in normal rats and in hypophysectomized rats treated for 24 h with GH or IGF-I using in situ hybridization and immunohistochemistry. Serum IGF-I, body weight, and tibial growth plate, but not articular cartilage, height increased with both treatments. Both IGF-I mRNA and IGF-I immunoreactivity occurred in all chondrocyte layers of growth plate and articular cartilage. The percentage of cells with IGF-I mRNA correlated well with IGF-I immunoreactivity under all experimental conditions. In normal rats, IGF-I expression was highest in the upper hypertrophic zone in growth plate (68-71%) and articular cartilage (32-34%). Hypophysectomy, GH, or IGF-I did not significantly affect this percentage. In the stem cell and proliferative and lower hypertrophic zones of growth plate, hypophysectomy dramatically reduced the percentage of labeled chondrocytes, and GH restored it. IGF-I increased IGF-I mRNA and immunoreactivity only in the proliferative zone. In articular cartilage, both remained unchanged under all experimental conditions. Together with our previous finding that GH infusion of hypophysectomized rats enhances chondrocyte maturation at all differentiation stages, the present results are compatible with the idea that IGF-I produced by all chondrocyte layers under the influence of GH mediates chondrocyte maturation and thus longitudinal bone growth in an autocrine/paracrine manner.     

Action of multiplication-stimulating activity on [3H]thymidine incorporation in rabbit and human fetal chondrocytes in vitro

The mitogenic action of multiplication-stimulating activity (MSA) on normal mammalian chondrocytes has been examined. Addition of MSA (NIH, PkII-MSA, 2.5-500 ng/ml or Collaborative Research, CR-MSA, 50-250 ng/ml) to primary suspensions of chondrocytes prepared by enzymic digestion of costal and articular cartilage of rabbits (356-481 g body wt) resulted in a dose-dependent increase in [3H]thymidine incorporation into the trichloroacetic acid-precipitated cell contents. CR-MSA (50-250 ng/ml) also had a significant stimulatory effect on [3H]thymidine incorporation into human fetal chondrocytes (22 weeks of gestation) prepared by enzymic digestion. When PkII-MSA was added in the presence of 1.25% of a standard adult or cord plasma to either rabbit or human fetal (18 weeks) chondrocytes, the increase in [3H]thymidine incorporation appeared to be synergistic. The mitogenic action of MSA can thus be demonstrated on primary suspensions of mammalian chondrocytes. The action of MSA on human chondrocytes has not previously been reported.     

Circulating growth factor studies in growth plate versus resting cartilage in vitro. II. Recognition of growth factors in rat serum

The growth-promoting effects of GH can be explained in part by the mediation of somatomedins/insulin-like growth factors (IGFs). However, large quantities of the IGFs are required to stimulate growth in vivo, and in some conditions, IGF levels may correlate poorly with GH levels and growth status. These observations suggest that other circulating factors may also be important for growth. We have examined the growth-promoting activity in rat serum, as assessed by stimulation of sulfate and/or thymidine uptake by resting and growth plate cartilage (osteochondral junction) from hypophysectomized rats in vitro. Although stimulation by a low molecular weight somatomedin fraction (approximately 5,000-12,000) accounted for about 90% of serum stimulation of sulfate uptake by resting cartilage, it explained only about 60% of stimulation of the growth plate. Growth plate and resting cartilage appeared equally insensitive to insulin, but the growth plate exhibited reduced sensitivity to inhibitor(s) in diabetic rat serum. Fractionation of normal rat serum by gel filtration at neutral pH revealed comparable stimulation of growth plate and resting cartilage by high molecular weight factors, presumably somatomedins bound to carrier proteins. After gel filtration at acid pH, both growth plate and resting cartilage responded to somatomedins with molecular weights from 5,000-12,000. However, the growth plate also responded to a 12,000-22,000 mol wt factor [Sephadex G-75; 5 X 120 cm; sulfate uptake, 68 +/- 16% above buffer (mean +/- SEM); P less than 0.01] which did not affect resting cartilage (sulfate uptake, 27 +/- 21% above buffer; P = NS). Levels of both the low and higher molecular weight factors were reduced in the serum of hypophysectomized rats. We conclude that circulating growth-promoting activity includes both the low molecular weight somatomedins and a higher molecular weight growth plate growth factor which is not recognized by resting cartilage. Use of the osteochondral junction assay system may permit elucidation of the regulation and nature of this growth factor.     

Effect of platelet lysate on growth and sulfated glycosaminoglycan synthesis in articular chondrocyte cultures

Human platelet lysate (PL) has a strong growth promoting action on rabbit articular chondrocytes in monolayer culture. The responsible factor is heat stable (56°C, 30 minutes) and above 10,000 MW. PL (80 μg protein/ml) reduces cell protein content and sulfated glycosaminoglycan synthesis. Synthesis of DNA is stimulated within the first 12 hours of culture but the decline in radiosulfact incorporation lags. PL acts to a slight extent on chondrocytes in serum-free media, but its effect is potentiated by “platelet-poor human serum” or fetal bovine serum. PL is one of several agents having such effects on chondrocytes cultured in serum-containing media. Stimulation of growth in this cell type thus reduces nonreplicative biosynthetic activity nonspecifically. In the epiphyseal growth plate and in pathologic alterations of permeability of the martix of articular cartilage, platelet-derived factors, together with somatomedin or other cofactors in serum, may be the principal mediator of growth of chondrocytes in vivo.     

Growth hormone induces multiplication of the slowly cycling germinal cells of the rat tibial growth plate.

To study the effect of locally infused growth hormone (GH) or insulin-like growth factor I(IGF-I) on slowly cycling cells in the germinal cell layer of the tibial growth plate, osmotic minipumps delivering 14.3 microCi of [3H]thymidine per day were implanted s.c. into hypophysectomized rats, and GH (1 microgram) or IGF-I (10 micrograms) was injected daily through a cannula implanted in the proximal tibia. The opposite leg served as a control. After 12 days of treatment, the osmotic minipumps were removed, and three rats in each group were given GH (20 micrograms/day, s.c.) for an additional 14 days to chase the labeled cells out of the proliferative layers. Labeled cells remained in the germinal layer, in the perichondrial ring, and on the surface of the articular cartilage close to the epiphyseal plate. GH administered together with labeled thymidine significantly increased the number of labeled cells in the germinal cell layer compared to that in the control leg (ratio = 1.95 +/- 0.13), whereas IGF-I showed no stimulatory effect (ratio = 0.96 +/- 0.04). Therefore GH but not IGF-I stimulates the multiplication of the slowly cycling (label-retaining) cells in the germinal layer of the epiphyseal plate. IGF-I acts only on the proliferation of the resulting chondrocytes.     

Effects of human and salmon calcitonin on human articular chondrocytes cultivated in clusters

The effects of different pharmacological concentrations (0, 5, 10, 100, and 1000 ng/mL) of synthetic human calcitonin (hCT) and salmon calcitonin (sCT) on the incorporation of [3H]thymidine and production of proteoglycans (PG) and type II collagen (coll II) by human articular chondrocytes during a 20-day period were studied in a tridimensional chondrocyte culture model. [3H]Thymidine uptake was measured in chondrocyte clusters, and specific PG and coll II RIAs were performed every 4 days on the culture medium and cell aggregates; total PG and coll II production were also assessed at different culture durations by adding the amounts found in culture media and their corresponding clusters. Incubation with hCT or sCT did not affect [3H]thymidine uptake regardless of the dose. For each culture period, PG and coll II release into culture medium, cluster content, and total production increased significantly in a dose-dependent manner. Cumulative curves for these parameters showed a progressive significant increase with culture duration at hCT and sCT doses of 0, 5, and 10 ng/mL. Cumulative curves obtained with 10, 100, and 1000 ng/mL were seldom significantly different from one another. No differences emerged between the use of hCT or sCT. Thus, CT exerted no proliferative effect on human articular chondrocytes in tridimensional culture, but displayed a dose-dependent and prolonged stimulatory effect on PG and coll II production. CT may possess chondroprotective properties in addition to its other known effects.     

Gremlin 1, Frizzled‐related protein,and Dkk‐1 are key regulators of human articular cartilage homeostasis

Objective

The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation.

Methods

Whole‐genome gene‐expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA.

Results

Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled‐related protein (FRP), and Dkk‐1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long‐bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single‐nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population‐based cohorts.

Conclusion

Taken together, our study identified Gremlin 1, FRP, and Dkk‐1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

     

Increased somatomedin and cartilage metabolic activity in rabbit fetuses injected with insulin in utero

Summary The effect of insulin injection in fetal rabbits on plasma somatomedin activity and cartilage metabolism was investigated. One fetus in each of 12 litters was injected with 1 unit of insulin zinc suspension subcutaneously on day 27 of gestation and a control fetus was injected with the same volume of 0.154 mol/l saline. The litter was delivered by caesarean section on day 29 and each fetus identified. Plasma somatomedin activity was determined by fetal rabbit cartilage bioassay. Costal cartilage from individual fetuses was incubated in medium containing [³H]thymidine or [³⁵S]sulphate as indicators of cell replication and matrix synthesis respectively. Individual values for somatomedin activity or cartilage isotope uptake were ranked within a litter. In each case the rank in the litter of the insulin-injected fetus, but not the saline-injected fetus, was significantly higher than the mean rank of the litter. Insulin did not stimulate cartilage metabolism in vitro.     

Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy.

OBJECTIVE: To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. METHODS. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. RESULTS. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. CONCLUSION. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.     

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.