Macrophage-derived soluble factors mediate suppression induced by 2,4-dinitrophenyl-conjugated mouse IgG in hybridoma cells

Our laboratory has established that 2,4-dinitrophenyl-conjugated mouse IgG (DNP-MGG) can specifically suppress proliferation, antibody synthesis, and secretion in vivo in two anti-DNP secreting cell lines: hybridoma 35-12 and myeloma MOPC-315. In the present study an in vitro system was used to further analyze the mechanism of suppression of hybridoma 35-12 cells (HC) by DNP-MGG. It was found that DNP-MGG-induced suppression of HC requires macrophages (M phi) and occurs only in eclipsed HC which are mainly small, nonsecreting cells. The M phi-mediated suppression is DNP specific, requires no M phi-HC cell contact, and does not involve killing of eclipsed HC. M phi culture supernatant alone cannot mediate suppression, but supernatants obtained by culturing M phi with either HC or supernatant from HC culture can mediate suppression of eclipsed HC in the presence of DNP-MGG. DNP-MGG is not required for the generation of effective M phi factors, but it is required for suppression of HC in the presence of M phi factors. Indomethacin cannot reverse M phi-mediated suppression, suggesting prostaglandins may not be the M phi factors. These data suggest that M phi-derived factors which are not prostaglandins in nature may play a role in B-cell regulation and in B-cell suppression induced by tolerogenic forms of antigen.     

Correlation of cell division and specific protein production during the course of lymphoid cell differentiation

Mice were immunized with horseradish peroxidase and injected with [³H] thymidine. Popliteal lymph node cells were submitted to electron microscopic immunocytochemistry and high resolution autoradiography in order to correlate antibody production and ability to undergo cell division at various stages of lymphoid cell differentiation. Antibody synthesis started in blast cells and increased steadily until the mature plasma cell stage was reached. Thymidine incorporation was highest in blastoid cells and decreased continuously afterwards. Chromatin dispersion was found to be paralleled by thymidine incorporation. This observation and data of other authors seem to indicate that chromatin dispersion is a prerequisite for replication. Almost all mature plasma cells were devoid of thymidine incorporation. This confirms that they are end cells apparently unable to divide.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

“Suppressor factor” of neutrophils: A short story of a long-term misconception

A large body of evidence obtained during the last decade has demonstrated that neutrophils suppress T cell proliferation in different models of inflammation and cell interaction. The commonly used method for assessing cell proliferation and proliferation inhibition is measuring [³H]thymidine incorporation into cells. Earlier, we observed inhibition of [³H]thymidine uptake in experiments on neutrophil-mediated regulation of T cell response in tuberculosis immunity. Here, we used different types of proliferating cells to analyze the nature of the soluble “neutrophil factor” by a variety of methods (dialysis, HPLC, mass spectrometry, and NMR) and unambiguously demonstrated that neutrophils do not synthesize a specific factor inhibiting cell proliferation, but secrete high concentrations of extracellular thymidine that competitively inhibit [³H]thymidine incorporation. Although the physiological significance of thymidine secretion by neutrophils remains unknown, this phenomenon should be carefully considered when designing test systems for studying cell–cell interactions.     

Reduced body fat and increased hepatic lipid synthesis in mice bearing interleukin-6-secreting tumor

Chronic secretion of interleukin-6 (IL-6) in mice causes metabolic alteration in the liver, leading to increased synthesis of hepatic cholesterol and fatty acids (FA). Mice were injected with allogeneic tumor cells transduced with the murine IL-6 gene. During the 3 wk after tumor inoculation, elevated serum IL-6 levels were associated with increased spleen and liver weight. Histological examination of sections from the liver showed increased hepatocyte proliferation, resulting in liver enlargement. Body composition analysis revealed that IL-6 caused a significant loss in fat tissue without affecting lean body mass and water content. Hepatic de novo synthesis of FA and cholesterol, as measured by (3)H(2)O incorporation, was three to five times as high in mice secreting IL-6 (IL-6 mice) as in pair-fed mice bearing nonsecreting tumors. This increase in FA and cholesterol synthesis is sufficient to maintain hepatic triglyceride secretion at levels comparable with those of pair-fed mice bearing nonsecreting tumors and, presumably, is the main source of cholesterol and FA-phospholipids necessary for hepatocyte proliferation.     

Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Mitogenicity and Adjuvanticity of a Marine Bacterium,Vibrio anguillarum,in Mice

The effects of whole cells of three different O serotypes of Vibrio anguillarum on the murine immune response were studied. The addition of different doses (1–100/ig/ml) of V. anguillarum cells, as well as Salmonella typhimurium lipopolysaccharide, markedly increased the incorporation of [³H] thymidine into in vitro cultured spleen cells of C57BL/6 mice. All three serotype strains of V. anguillarum were able to induce the mitogenic effect at 10 μg/ml and 100 μg/ml, but serotype I strains were more potent than the others. Since pretreatment of spleen cells with rabbit anti-mouse thymocyte antiserum did not affect the mitogenic activity of V. anguillarum, Vibrio cells may be a B-lymphocyte mitogen. When sheep or horse erythrocytes and Vibrio cells were injected intraperitoneally into ddY mice, Vibrio cells exhibited an enhancing effect on antibody response in vivo, regardless of the different serotypes. Vibrio cells, when injected intraperitoneally into mice before the antigen, markedly suppressed the antibody response. Several days after the injection of Vibrio cells, these mice showed an enhanced carbon clearance activity. Acid phosphatase activity in their peritoneal cells was also augmented, suggesting that Vibrio cells activated macrophages in the mice.     

Enhancing and suppressive effects of macrophages on T-lymphocyte stimulation in vitro.

The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [¹²⁵I]UdR incorporation as a measure of lymphocyte proliferation. [¹²⁵I]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers of peritoneal macrophages beyond 5–10%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9–10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [¹²⁵I]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.     

Synthesis of ubiquinone and cholesterol in human fibroblasts: regulation of a branched pathway.

The current studies demonstrate that cultured human flbroblasts utilize mevalonate for the synthesis of ubiquinone-10 as well as for the synthesis of cholesterol. Study of the regulation of this branched pathway was facilitated by incubating the cells with compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryI coenzyme A reductase, which blocked the formation of mevalonate within the cell. The addition of known amounts of [³H]mevalonate to the culture medium in the presence of compactin permitted the study of the relative rates of mevalonate incorporation into cholesterol and ubiquinone-10 under controlled conditions. When low concentrations of exogenous [³H]mevalonate (10 to 50 μm) were added to cells that were provided with exogenous cholesterol in the form of plasma low density lipoprotein (LDL), the cells incorporated the [³H]mevalonate into ubiquinone-10 at a rate that was two- to threefold faster than the incorporation into cholesterol. When the cells were deprived of exogenous LDL-cholesterol, the incorporation of [³H]mevalonate into ubiquinone-10 decreased and the incorporation of [³H]mevalonate into cholesterol increased. As a result, in the absence of exogenous cholesterol more than 60 times as much [³H]mevalonate was incorporated into cholesterol as into ubiquinone-10. Considered together with previous findings, the current data are compatible with a regulatory mechanism in which LDL inhibits cholesterol synthesis in fibroblasts at two points: (1) at the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, thereby inhibiting mevalonate synthesis, and (2) at one or more points distal to the last intermediate common to the cholesterol and ubiquinone-10 biosynthetic pathways. The latter inhibition allows ubiquinone-10 synthesis to continue in the presence of LDL despite a 98% reduction in mevalonate synthesis.     

Induction of tumor-specific acquired immunity against already established tumors by selective stimulation of innate DEC-205+ dendritic cells

Two major distinct subsets of dendritic cells (DCs) are arranged to regulate our immune responses in vivo; 33D1⁺ and DEC-205⁺ DCs. Using anti-33D1-specific monoclonal antibody, 33D1⁺ DCs were successfully depleted from C57BL/6 mice. When 33D1⁺ DC-depleted mice were stimulated with LPS, serum IL-12, but not IL-10 secretion that may be mediated by the remaining DEC-205⁺ DCs was markedly enhanced, which may induce Th1 dominancy upon TLR signaling. The 33D1⁺ DC-depleted mice, implanted with syngeneic Hepa1-6 hepatoma or B16-F10 melanoma cells into the dermis, showed apparent inhibition of already established tumor growth in vivo when they were subcutaneously (sc) injected once or twice with LPS after tumor implantation. Moreover, the development of lung metastasis of B16-F10 melanoma cells injected intravenously was also suppressed when 33D1⁺ DC-deleted mice were stimulated twice with LPS in a similar manner, in which the actual cell number of NK1.1⁺CD3⁻ NK cells in lung tissues was markedly increased. Furthermore, intraperitoneal (ip) administration of a very small amount of melphalan (l-phenylalanine mustard; l-PAM) (0.25 mg/kg) in LPS-stimulated 33D1⁺ DC-deleted mice helped to induce H-2K^b-restricted epitope-specific CD8⁺ cytotoxic T lymphocytes (CTLs) among tumor-infiltrating lymphocytes against already established syngeneic E.G7-OVA lymphoma. These findings indicate the importance and effectiveness of selective targeting of a specific subset of DCs, such as DEC-205⁺ DCs alone or with a very small amount of anticancer drugs to activate both CD8⁺ CTLs and NK effectors without externally added tumor antigen stimulation in vivo and provide a new direction for tumor immunotherapy.     

Suppression of IgE antibody production in SJL mice. V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram DNP-Nb and 1 mg A1(OH)3. The immunized SJL mice were irradiated (60-540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Scintigraphic detection of xenografted tumors producing human basic fibroblast growth factor

A murine monoclonal antibody 3H3 recognizes the basic fibroblast growth factor (FGF) and inhibits the growth of human glioblastoma cells both in vitro and in vivo. We studied the potential of a scintigraphic technique using the 3H3 antibody to detect tumors that produce basic FGF.¹²⁵I- and¹¹¹In-labeled 3H3 bound to U87MG human glioblastoma cells in vitro. U87MG cells were inoculated subcutaneously into nude mice. After development of the tumor, radiolabeled 3H3 was injected into the subcutaneous space surrounding the tumor. A high level of radioactivity from 3H3 was retained at the tumor, whereas an irrelevant antibody cleared rapidly from the injected site. Radiolabeled 3H3 was not retained in tumors that did not produce basic FGF. Scintigraphic detection of tumors expressing basic FGF would be valuable for the therapeutic application of the antibody.     

Distinctions between rabbit lymph node cell populations responding to primary and secondary injections of keyhole limpet hemocyanin

Rabbits were injected once or twice into the hind foot pads with alum-precipitated keyhole limpet hemocyanin. At the height of the primary or secondary responses individual rabbits were sacrificed for the preparation of lymph node cell suspensions from the regional lymph nodes. These cells were employed for the in vitro study of antibody synthesis by the incorporation of ¹⁴C-leucine and the secretion of antibody by the time of appearance of radioactive antibody in the medium. The primary response cells rapidly synthesized and secreted IgM and IgG antibodies. The secondary response cells rapidly synthesized IgM and IgG antibodies, secreted IgG antibody promptly, but secreted the IgM antibody with a lag of six to ten hours. Microsomal fractions could not be prepared from the primary response cells, but were readily produced from the cells of the secondary response. The primary response cells contained mainly free ribosomes, those of the secondary response predominantly membrane-bound ribosomes. It was postulated that IgM antibody was not secreted until it was glyco-sylated in the Golgi apparatus and that the lag in secretion entailed the time for this rate-limiting step to occur.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.     

Polyamine-dependent alterations in the structure of microfilaments,golgi apparatus,endoplasmic reticulum,and proteoglycan synthesis in BHK cells

The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a β-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [³H]glucosamine and [³⁵S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation. J. Cell Biochem. 66:165-174, 1997. © 1997 Wiley-Liss, Inc.     

DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

To investigate the response of cells to one type of DNA damage — namely DNA crosslinks — cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 μg/ml plus 5 kJ/m² or 1.0 μg/ml plus 1–2.5 kJ/m²)_halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [³H]thymidine into cellular DNA immediately after the treatment. Then, after 8 h of incubation, the incorporation of [³H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [³]Hthymidine were correlated with the halt and resumption in the cell-cycle process.Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.