Therapeutic touch stimulates the proliferation of human cells in culture.

OBJECTIVES: Our objective was to assess the effect of Therapeutic Touch (TT) on the proliferation of normal human cells in culture compared to sham and no treatment. Several proliferation techniques were used to confirm the results, and the effect of multiple 10-minute TT treatments was studied. DESIGN: Fibroblasts, tendon cells (tenocytes), and bone cells (osteoblasts) were treated with TT, sham, or untreated for 2 weeks, and then assessed for [(3)H]-thymidine incorporation into the DNA, and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). The number of PCNA-stained cells was also quantified. For 1 and 2 weeks, varying numbers of 10-minute TT treatments were administered to each cell type to determine whether there was a dose-dependent effect. RESULTS: TT administered twice a week for 2 weeks significantly stimulated proliferation of fibroblasts, tenocytes, and osteoblasts in culture (p = 0.04, 0.01, and 0.01, respectively) compared to untreated control. These data were confirmed by PCNA immunocytochemistry. In the same experiments, sham healer treatment was not significantly different from the untreated cultures in any group, and was significantly less than TT treatment in fibroblast and tenocyte cultures. In 1-week studies involving the administration of multiple 10-minute TT treatments, four and five applications significantly increased [(3)H]-thymidine incorporation in fibroblasts and tenocytes, respectively, but not in osteoblasts. With different doses of TT for 2 weeks, two 10-minute TT treatments per week significantly stimulated proliferation in all cell types. Osteoblasts also responded to four treatments per week with a significant increase in proliferation. Additional TT treatments (five per week for 2 weeks) were not effective in eliciting increased proliferation compared to control in any cell type. CONCLUSIONS: A specific pattern of TT treatment produced a significant increase in proliferation of fibro-blasts, osteoblasts, and tenocytes in culture. Therefore, TT may affect normal cells by stimulating cell proliferation.

Differential effects of ellipticine and aza-analogue derivatives on cell cycle progression and survival of BALB/c 3T3 cells released from serum starvation or thymidine double block.

10-[Diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5] pyrrolo[2,3-g]isoquinoline (BD-40) (NSC-327471D) is an aza-ellipticine derivative with a promising antitumor activity (M. Marty, C. Jasmin, P. Pouillard, C. Gisselbrecht, G. Gouvenia, and H. Magdalainat, 17th Annual Meeting of the American Society of Clinical Oncology, C-108, 1981) and less toxicity than ellipticine. We have compared the effects of ellipticine, several of its analogues, and two aza-analogue ellipticine derivatives (BD-40 and BR-1376) on cell cycle progression of BALB/c 3T3 mouse cells under different growth conditions. Both drug series were found to stop cell growth and block cells in G2 phase in exponentially growing cultures and cultures released from a thymidine double block. Long-term viability of these cells was completely suppressed after a short exposure to the drugs. In contrast, while ellipticine and its derivatives caused identical effects in cells recovering from serum starvation, BD-40 and BR-1376 did not block cells in G2 phase and did not prevent the completion of the first division round occurring after serum addition to quiescent cells. This transient refractory state was accompanied by a total conservation of long-term viability of these cells at least for the next 6 h following serum and drug addition. This lack of effect was not related to an impaired drug uptake by cells recovering from serum starvation or by a dramatic change in drug distribution inside the cells. These results indicate that the nitrogen substitution in the ellipticine heterocycle is an important if not unique feature for the particular effect of the aza-analogues of ellipticine. Furthermore, they suggest that, in contrast to ellipticine derivatives, these compounds require an activation step before exhibiting cytotoxicity.

Arginase, an S-phase enzyme in a human cell line.

Suspension cultures of 'Chang liver' cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S + G2) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G2/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in arginase activity during the mitotic cycle were the result of variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4--6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in 'Chang liver' cells is discussed.

Evidence for the involvement of endogenous thymidine in the density-inhibition of tumorigenic Chinese hamster V79 cells.

Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell line. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient (TK-) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK- or TK-proficient (TK+) cells was examined. As a result, the growth of TK- cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK+ cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the supernatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in confluent culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant growth in the presence of certain growth inhibitors produced by density-inhibited V79 cells.

Antitumor activity of ZD1694 (tomudex) against human head and neck cancer in nude mouse models: role of dosing schedule and plasma thymidine.

We studied the antitumor activity and toxicity of ZD1694 (tomudex), a specific inhibitor of thymidylate synthase (TS), in nude mice bearing human head and neck squamous cell carcinoma A253 and FaDu xenografts. Mice were treated by single i.v. push (i.v. x 1), i.v. push once a week for 3 weeks (weekly x 3), and i.v. push once a day for 5 days (daily x 5), and the maximum tolerated doses (MTDs) of ZD1694 were 300 mg/kg, 60 mg/kg/week, and 30 mg/kg/day, respectively. ZD1694 was moderately active against both A253 and FaDu xenografts. Antitumor activity was schedule-dependent in both tumors: weekly x 3 > or = i.v. x 1 >> daily x 5. In contrast, the rank order of toxicity was daily x 5 >> weekly x 3 > or = i.v. x 1. ZD1694 at the MTD produced 20% complete tumor regression and 20% partial tumor regression (PR) with i.v. x 1 and weekly x 3 schedules and 12-day tumor growth delay with daily x 5 schedule against FaDu xenografts. No complete tumor regression was achieved with ZD1694 with any schedule against A253; a 20% PR, 40% PR, and 10-day tumor growth delay were observed with i.v. x 1, weekly x 3, and daily x 5 schedules, respectively. The data indicate that ZD1694 was slightly more effective against FaDu than against A253. Of interest and potential clinical importance was the observation that ZD1694 was still active at doses lower than the MTD (> or =1/3 MTD), which showed a high therapeutic index and wide safety margin. Study of ZD1694 compared with 5-fluorouracil and 5-fluoro-2'-deoxyuridine at the MTD revealed that the antitumor activity of ZD1694 was comparable with or superior to 5-fluorouracil and 5-fluoro-2'-deoxyuridine against both A253 and FaDu xenografts, with less toxicity. High plasma thymidine in mouse relative to human (approximately 1.3 microM and <0.1 microM, respectively) may complicate the study of antitumor activity and toxicity of TS inhibitors with human tumor xenografts grown in the mouse. To test this hypothesis, we preadministered methoxypolyethyleneglycol-conjugated thymidine phosphorylase (MPEG-TPase; 2500 units/kg/dose) to reduce mouse plasma thymidine, then treated with various doses of ZD1694 using the daily x 5 or i.v. x 1 schedules in the A253 tumor model. MPEG-TPase significantly increased the toxicity of ZD1694; the MTD of ZD1694 plus MPEG-TPase was reduced 3- and 10-fold compared with ZD1694 alone for i.v x 1 and daily x 5 schedules, respectively. However, preadministration of MPEG-TPase did not potentiate the antitumor activity of ZD1694 with either schedule. The data indicate that the study of TS inhibitors in rodent models may not be suitable for predicting a safe dose for clinical study. However, rodent models, particularly human tumor xenografts, are still useful models for evaluation of antitumor activity and schedule selection for TS inhibitors.

AT islands - their nature and potential for anticancer strategies.

The human genome contains a unique class of domains, referred to as AT islands, which consist typically of 200-1000 bp long tracts of up to 100% A/T DNA. The significance of AT islands as potential targets for chemotherapeutic intervention stems from two main aspects. First, AT islands are inherently unstable (expandable) minisatellites that are found in various known loci of genomic instability, such as AT-rich fragile sites. Second, AT islands are involved in the organization of the genomic DNA on the nuclear matrix by acting as scaffold/matrix attachment regions, S/MARs. DNA duplexes of AT islands are unusually flexible and prone to base unpairing, which are crucial MAR attributes. Various AT islands show high binding affinity for isolated nuclear matrices and associate with the nuclear matrix in the cell. The cellular MAR function of AT islands may differ in cancer and normal cells. The abnormally expanded AT islands in the FRA16B fragile site in leukemic CEM cells act as strong, permanent MARs, while their unexpanded counterparts in normal cells are loop localized. Given their instability and involvement in the remodeling of the nuclear architecture, AT islands may be a factor in cancerous phenotypes. AT islands are preferentially targeted by the extremely potent DNA-alkylating antitumor drugs, bizelesin and U78779. High lethality of lesions in AT islands is consistent with the critical role of MAR domains in DNA replication. The abnormal structure/function of AT islands, such as their expansion and acquired strong MAR properties, may sensitize cancer cells to AT island targeting drugs.

Role of hypoxanthine and thymidine in determining methotrexate plus dipyridamole cytotoxicity.

The nucleoside transport inhibitor dipyridamole can potentiate the cytotoxicity of methotrexate by a mechanism that was thought to be related to the inhibition of thymidine salvage. In human ovarian carcinoma cells thymidine only partly reversed the in vitro cytotoxicity of methotrexate plus dipyridamole at sub-millimolar concentrations, above which the cytotoxicity of thymidine itself became evident. Hypoxathine with thymidine, or hypoxanthine alone at a higher concentration, completely reversed methotrexate and methotrexate plus dipyridamole cytotoxicity. The effects of dipyridamole on cellular cyclic adenosine monophosphate (cAMP) levels and on 3H-methotrexate efflux in 2008 cells were examined. At 10 mumol/l, dipyridamole did not alter cAMP content or methotrexate influx in ovarian carcinoma cells, but reduced the rate of efflux of 3H-methotrexate by 25%. In Chinese hamster ovary cells and their folylpolyglutamyl synthase-deficient variant AUX B1, the reduced methotrexate efflux by dipyridamole was not due to increased polyglutamation, since increased retention was observed in both cell lines. The data support the hypothesis that dipyridamole potentiated the activity of methotrexate by inhibiting the salvage of hypoxanthine, and to a lesser extent, that of thymidine. The ability of dipyridamole to increase the cellular retention of methotrexate was probably a non-specific action of dipyridamole on the cell membrane, and may have a role in the observed synergy.

Flow cytometric analysis of pig epidermal keratinocytes: effects of ultraviolet B irradiation (UVB) and topical PUVA treatment.

The effects of a single application of ultraviolet B irradiation (UVB) and topical PUVA treatment on pig epidermal cell kinetics were studied by DNA-flow cytometry (FCM), 3H-thymidine uptake, mitotic counts and 2-3H-deoxy-D-glucose uptake. Following UVB irradiation (2MED: 250 mJ/cm2) and PUVA (0.9, 1.4 J/cm2) treatment, thymidine uptake and mitosis were markedly decreased. This was followed by a transient increase in all of these parameters. The maximal increase was observed at 96 h following the UVB irradiation and at 168 h following the PUVA treatment (0.9 J/cm2), respectively. The suppression of DNA synthesis and mitosis persisted for a longer period in PUVA-treated than in UVB-treated epidermis. At 48-72 h after the UVB irradiation and 72-144 h after the PUVA treatment, an increase in the cells of the G2/M fraction was observed. This was associated with the decreased mitotic counts, suggesting accumulation of G2-blocked cells. Histologically, PUVA-treated epidermis showed a considerable degenerative change. Mild acanthosis was noted at 72-96 h in UVB-treated epidermis and at 168 h in PUVA-treated epidermis. These results indicate that the inhibition of DNA synthesis and increase in G2-phase cells are associated with the UVB and PUVA induced suppression of epidermal cell proliferation. These suppressive effects that persisted longer in PUVA-treated, than in UVB-treated epidermis, were followed by an increased epidermal keratinocyte proliferation of pig skin in vivo.

Linear-quadratic analysis of radiosensitization by halogenated pyrimidines. I. Radiosensitization of human colon cancer cells by iododeoxyuridine.

Radiosensitization by iododeoxyuridine (IdU) is a method of enhancing cell killing in the radiotherapy of human cancers, especially for tumors that proliferate faster than the surrounding normal tissues, such as might appear in brain or liver. We have investigated in vitro the relationship between the amount of thymidine replacement by IdU and the resulting radiosensitization in two human colon cancer cell lines, HCT 116 and HT 29, with differing inherent sensitivities to X rays. The results show that an increase in the initial slope of the cell survival curve was the predominant mode of radiosensitization. In this situation, the emphasis on changes in the initial slope suggest the use of a survival curve model that contains the initial slope as a defined variable, which the traditional single-hit, multitarget model does not. We present our analyses mainly in terms of alpha (initial slope) and changes in surviving fraction at 2 Gy and also as a modified form of sensitizer enhancement ratio that describes the dose-modifying factor of IdU at a single radiation dose of 2 Gy (SER 2 Gy). Iododeoxyuridine is an effective radiosensitizer in both cell lines, but IdU appears especially effective in increasing the initial slope of the more radioresistant line, the HT 29 cells.

Linear-quadratic analysis of radiosensitization by halogenated pyrimidines. II. Radiosensitization of human colon cancer cells by bromodeoxyuridine.

As a continuation of the studies in Part I (Miller, Fowler, and Kinsella, Radiat. Res. 131, 000-000, 1992), which examined the radiosensitizing effects of iododeoxyuridine (IdU), similar experiments with bromodeoxyuridine (BrdU) were conducted concurrently to characterize its effects on the shape of the radiation survival curves of cells of two human colon cancer cell lines, HT 29 and HCT 116. The efficiency of radiosensitization by BrdU, expressed as a function of percentage thymidine replacement, was lower when compared to IdU in both cell lines. However, the major radiosensitizing effect of BrdU was manifest as an increase in the initial slope (alpha), just as observed for IdU. However, with BrdU, in contrast to IdU, an increase in curvature (repairable damage) was also evident. Cells of the more radiosensitive line, HCT 116, showed less sensitization by either BrdU or IdU than cells of the more radioresistant line, HT 29. These results were consistent with the proposed mechanism of radiosensitization being an increase in the single-hit character of low-LET radiation. It follows that the radiosensitizing effects of both analogs were largest in the low-dose region of the survival curve.

Normal human granulopoiesis revisited. I. Blood data.

The absence of an unquestionable label to study the components of normal granulopoiesis leads experimentalists to design and develop numerous different techniques. An extensive review of the corresponding human data is performed concerning blood granulocyte cellularity, sojourn time and turnover rate, bone marrow neutrophil cellularity, production, mitotic activities, kinetic parameters and differential cell counts. Advantages and pitfalls relevant to each experimental procedure are discussed. Results obtained from recently devised experimental designs are included and compared to the results derived from more classical techniques. In particular data concerning blood granulocytes obtained through the in vitro labelling procedure using either diisopropylfluorophosphate or radiochromate are re-visited in the light of recently published results derived from techniques using a pulse or a continuous infusion of tritiated thymidine. Although some questions remain, particularly concerning bone marrow differential cell counts, reference data are proposed to elaborate a complete and coherent scheme of normal human neutrophilic granulopoiesis.

[Cell death and genetic alterations induced by thymidylate stress].

Thymidylate synthase-negative mutants of mouse FM3A cells are immediately committed to cell death upon thymidine starvation. Thymineless death can be mimicked by use of drugs that block the formation of reduced folate or dTMP. In parallel with this thymineless death, DNA double strand breaks and chromosome aberrations are induced. Thymidylate starvation does not induce mutation, whereas very mild thymidylate starvation does. Thymidylate starvation, however, induces strongly a deletion of a heterologous genetic marker at specific sites which is stably integrated in a mouse cell chromosome by means of DNA-mediated gene transfer.

Human RECQL5 overcomes thymidine-induced replication stress.

Accurate DNA replication is essential to genome integrity and is controlled by five human RecQ helicases, of which at least three prevent cancer and ageing. Here, we have studied the role of RECQL5, which is the least characterised of the five human RecQ helicases. We demonstrate that overexpressed RECQL5 promotes survival during thymidine-induced slowing of replication forks in human cells. The RECQL5 protein relocates specifically to stalled replication forks and suppresses thymidine-induced RPA foci, CHK1 signalling, homologous recombination and gammaH2AX activation. It is unlikely that RECQL5 promotes survival through translesion synthesis as PCNA ubiquitylation is also reduced. Interestingly, we also found that overexpressing RECQL5 relieves cells of the cell cycle arrest normally imposed by thymidine, but without causing mutations. In conclusion, we propose that RECQL5 stabilises the replication fork allowing replication to overcome the effects of thymidine and complete the cell cycle.

Thymidine-dependent Staphylococcus aureus small-colony variants are associated with extensive alterations in regulator and virulence gene expression profiles.

Chronic airway infection is a hallmark of cystic fibrosis (CF) and many CF patients are infected persistently by Staphylococcus aureus. Thymidine-dependent trimethoprim-sulfamethoxazole (SXT)-resistant S. aureus small-colony variants (SCVs), often in combination with isogenic normal S. aureus phenotypes, are highly prevalent and persistent in airway secretions of CF patients due to long-term SXT therapy (B. Kahl, M. Herrmann, A. S. Everding, H. G. Koch, K. Becker, E. Harms, R. A. Proctor, and G. Peters, J. Infect. Dis. 177:1023-1029, 1998). In this report, SCVs were compared to normal S. aureus by transcription analysis of important regulator (sigB, sarA, and agr) and virulence (alpha-hemolysin, hla, and protein A, spa) genes. Growth curve analyses revealed longer doubling times and lower final densities for SCVs than for normal strains. sigB activity was measured by transcription analysis of the sigB target gene asp23. For nearly all SCVs, expression of all regulators was decreased as assessed by asp23 reverse transcription-PCR for sigB and Northern analysis for sarA and agr. These results are in agreement with diminished hla signals in all SCVs and increased spa signals in 5 of 10 SCVs compared to the isogenic normal S. aureus. Both supplementation of SCVs with thymidine and activation of the agr quorum-sensing system by the supernatant of the isogenic normal strain reversed transcription to almost normal levels. In conclusion, multiple changes in growth characteristics and in regulator and virulence gene expression render SCVs less virulent and allow them to survive in the hostile environment present in the airways of CF patients, thereby illustrating adaptation of the bacteria during long-term persistence.

Combined autoradiography and histochemistry: the simultaneous detection of 6-3H thymidine and acid phosphatase activity in cryostate sections.

A method is described for the simultaneous demonstration of 6-3H thymidine incorporation and acid phosphatase activity in cryostae sections of mouse thymus. The method enables a comparison of mitosis and acid hydrolase activity to be made in the same tissue section. In 8-week-old mice acid phosphatase positive cells reprsent 1.23 +/- 0.06% of the total population and 8.4 +/- 0.27% of the cells incorporate tritiated thymidine. Acid phosphatase activity can be used to estimate cell autolysis and death. The implication of the method in relation to tissue dynamics is discussed.

Mouse cytolytic T lymphocytes induced by xenogeneic rat stimulator cells exhibit specificity for H-2 complex alloantigens.

When mouse spleen cells were stimulated with irradiated xenogeneic, allogeneic, or trinitrophenyl-modified syngeneic lymphoid cells, the strongest cytolytic response was induced by alloantigens. Mouse cytolytic T lymphocytes generated to rat lymphoid cells demonstrated specificity for the immunizing rat strain, but extensive lysis of allogeneic target cells from certain mouse strains was also observed. Cold target inhibition studies indicated that separate clones of xenoantigen-induced cytolytic T lymphocytes lysed each of the allogeneic murine targets. [3H]Thymidine suicide of the effector cells generated to the rat stimulators revealed that only some of all potentially reactive mouse cytolytic T lymphocyte precursors with specificity for a given allogeneic target are activated by the stimulation with rat cells. This evidence that xenoantigens induce alloreactive cytolytic T lymphocyte receptor repertoire is directed at variants of autologous major histocompatibility complex antigens.

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions.

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).

Long-distance radical cation migration in duplex DNA: the effect of contiguous A.A and T.T mismatches on efficiency and mechanism.

A series of DNA oligomers was prepared. Each oligomer contained an anthraquinone group (AQ, sensitizer) covalently linked at a 5'-end and two GG steps that surrounded a variable region. The variable region was composed of A.T base pairs or A.A or T.T mismatches. Irradiation of the AQ injected a radical cation (hole) into the DNA that migrated through the duplex, being trapped by reaction with H2O of O2 at the GG steps. The effect of substituting A.A or T.T mismatches for Watson-Crick base pairs was examined. For A.A mispairs, charge transfer through the mismatch region was as efficient as through normal DNA. For the T.T mismatches, radical cation transport was strongly distance-dependent. These findings suggest that A.A mismatches form a zipper-like motif, and charge transport proceeds by a hopping mechanism. In contrast, charge transport through the T.T mismatches (where there are no purines) may proceed by quantum mechanical tunneling.

Parietal cell density during gastric ulcer healing in the rat.

BACKGROUND: During the healing of gastric ulcers, extensive changes take place in the structure and proportion of the parietal cells in the ulcer margin. We aimed to determine whether these changes are influenced by compounds or by procedures that may promote or delay gastric ulcer healing. METHODS: Acetic acid ulcers were produced in the rat gastric corpus; control rats were non-operated or sham-operated. All rats were provided with an osmotic minipump releasing 3H-thymidine for determination of the labelling index (LI). The rats were given omeprazole, gastrin or indomethacin, infected with Helicobacter pylori, or subjected to anterior vagotomy. They were killed after 1, 2, 6 or 13 days, and the ulcer margin and undamaged corpus wall were excised for histology. The proportion of parietal cells was calculated in relation to the total number, and the total volume, of the epithelial cells. RESULTS: The parietal cells averaged 16%-21% of the number and 30%-32% of the volume of the epithelial cells in the unoperated control rats, but considerably less in the ulcer margin. This reduction was partially prevented by indomethacin. In the undamaged, dorsal epithelium the parietal cells increased to 23%-26% of the number, and 41%-47% of the volume, of the epithelial cells, both in the ulcer rats and in the sham-operated rats. The LI of the parietal cells was 6% in the undamaged epithelium of the 13-day ulcer rats, but close to 0% in all other groups examined. CONCLUSION: Indomethacin prevents much of the loss of parietal cells in the ulcer margin. In the undamaged epithelium of the ulcer rats there is an increased proportion of parietal cells. The new parietal cells are not formed directly from dividing cells, but are probably recruited from existing precursor cells.

Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.