Enhanced DNA Synthesis Research Articles

Effects of albumin-bilirubin complexes with syngeneic or allogeneic albumin on DNA and protein synthesis in liver and spleen of partially hepatectomized rats

The effects of non-covalently bound complexes of allogeneic or syngeneic albumin with bilirubin and of albumin alone on DNA and protein synthesis in rat liver and spleen cells after partial hepatectomy were studied. The assay procedure was based on different intravenous doses of these compounds in rats after partial hepatectomy. The allogeneic albumin-bilirubin complex (at protein doses of 0.9 and 90 micrograms/100 g body weight) stimulated DNA and protein synthesis in liver cells irrespective of the dose. At a dose of 0.9 micrograms the syngeneic albumin-bilirubin complex enhanced DNA synthesis insignificantly and produced no effect on protein synthesis, while at a dose of 90 micrograms, both DNA and protein synthesis were considerably increased. Allogeneic or syngeneic albumin at the above doses stimulated only protein, not DNA, synthesis in the liver, while the highest stimulation was at 90 micrograms allogeneic albumin. It was found also that partial hepatectomy decreased DNA and protein synthesis in spleen cells. Albumin-bilirubin complex with allogeneic or syngeneic albumin and albumin alone either significantly enhanced DNA and protein synthesis in the spleen, compared to controls, or only restored synthesis to control levels. Thus DNA and protein synthesis in the regenerating liver and spleen was significantly enhanced after the injection of small doses of the albumin-bilirubin complex, indicating the existence of small amounts of a similar endogenous complex in the blood stream.

Deletion of exon 21 of the insulin receptor eliminates tyrosine kinase activity but preserves mitogenic signaling

To study the function of exon 21 of the insulin receptor, a mutant human insulin receptor lacking this domain was constructed. The mutant HIR delta E21 cDNA was transfected into Rat-1 fibroblasts and stable cell lines were selected. The HIR delta E21 receptors were expressed on the cell surface, and they bound insulin with the same affinity as did the wild-type-expressing cell line, hIRcB. The HIR delta E21 receptors did not display detectable autophosphorylation or kinase activity, and as expected, internalization was impaired and metabolic signaling properties were absent. Unexpectedly, insulin's ability to stimulate DNA synthesis in cells expressing HIR delta E21 receptors was far greater than that in the parental Rat-1 cells and equal to that measured in the hIRcB cell line. The enhanced mitogenic signaling properties of the HIR delta E21 receptors was confirmed by showing that treatment of HIR delta E21 cells with a human-specific insulin-mimetic anti-insulin receptor antibody also led to enhanced DNA synthesis. Thus, although no insulin receptor autophosphorylation or kinase activity was detectable in HIR delta E21 cells, these cells displayed enhanced insulin-induced mitogenic signaling. These results suggest that an alternative non-kinase-dependent stimulus-response pathway exists for the long-term biological effects of insulin.

Basic fibroblast growth factor may mediate proliferation in the compensatory adrenal growth response.

We have investigated the role of basic fibroblast growth factor (bFGF) in the neurally mediated compensatory adrenal growth response. Unilateral adrenalectomy resulted in a 13, 6, and 22% increase in adrenal weight, protein, and DNA content, respectively, and 33-40% increases in the rate of cell proliferation measured by [3H]thymidine incorporation in vitro. Three forms of bFGF, approximately 18.6, 21, and 22.5 kDa, were identified in rat adrenals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. bFGF was localized immunocytochemically in cells of the glomerulosa and the medulla. bFGF stimulated a 68-80% increase in the rate of DNA synthesis in adrenal capsule-glomerulosa preparations in vitro. Suramin (0.1 mM), a growth factor antagonist, blocked bFGF receptor interaction in vitro and, at 200 mg/kg given 5-7 days before adrenal surgery, blocked compensatory growth. Conversely, at 2.0 mg/kg, suramin significantly enhanced the compensatory growth response, perhaps caused by suramin-induced bFGF receptor upregulation, since suramin pretreatment also enhanced DNA synthesis in response to exogenous bFGF in vitro. These results suggest that bFGF may mediate proliferation in the compensatory adrenal growth response.

Artemisinin and Its Derivatives Enhance T Lymphocyte-Mediated Immune Responses in Normal Mice and Accelerate Immunoreconstitution of Mice with Syngeneic Bone Marrow Transplantation

Artemisinin and its derivatives, dihydroartemisinin and sodium artesunate, enhanced DNA synthesis of mouse spleen cells that had been activated with alloantigens or T cell mitogen Con A, but not B cell mitogen LPS, both in vitro and ex vivo. In vivo experiments showed that DTH response and antibody response against sheep erythrocytes were augmented in sodium artesunate-treated animals. In addition, sodium artesunate, a representative of these compounds, increased IL-2 production from mouse splenocytes stimulated with Con A. Because of the long-term T cell function deficiency in mice after bone marrow transplantation, the effect of sodium artesunate on the immune reconstitution was assessed. The results demonstrated that 10 mg/kg sodium artesunate significantly accelerated the immune reconstitution in mice after syngeneic bone marrow transplantation. These data suggested that artemisinin and its derivatives appeared to promote T cell function selectively, and these compounds had a potential application for the recovery of immune function.

Effects of arsenic on DNA synthesis in human lymphocytes.

Effects of arsenic on DNA synthesis in human lymphocytes were biphasic: Either trivalent (arsenic trioxide and sodium arsenite) or pentavalent (sodium arsenate) arsenic compounds at very low concentrations enhanced DNA synthesis in human lymphocytes stimulated by phytohemagglutinin (PHA), whereas higher concentrations inhibited DNA synthesis. There were differences among individual susceptibilities to arsenic-induced DNA synthesis. Either stimulating or inhibiting effects of trivalent arsenic on DNA synthesis in PHA-stimulated lymphocytes were always stronger than those of pentavalent arsenic. Both trivalent and pentavalent arsenic could be rapidly taken up into the human lymphocytes and immediately stimulate or inhibit DNA synthesis. A possible dual effect of arsenic at very low concentrations as both comutagen and inhibitor of mutagenesis is discussed.

Effects of arsenic on DNA synthesis in human lymphocytes stimulated by phytohemagglutinin.

Effects of arsenic on DNA synthesis in human lymphocytes were biphasic: either trivalent (arsenic trioxide and sodium arsenite) or pentavalent (sodium arsenate) arsenic compounds at very low concentrations enhanced DNA synthesis in human lymphocytes stimulated by phytohemagglutinin (PHA), whereas higher concentrations inhibited DNA synthesis. There were differences among individual susceptibilities to arsenic-induced DNA synthesis. Either stimulating or inhibiting effects of trivalent arsenic on DNA synthesis in PHA-stimulated lymphocytes were always stronger than those of pentavalent arsenic. It was also shown that both trivalent and pentavalent arsenic could be rapidly taken up into the human lymphocytes, and immediately stimulated or inhibited DNA synthesis. A possible dual effect of arsenic at very low concentrations as both comutagen and inhibitor of mutagenesis is discussed.

Analysis of myeloid characteristics in acute lymphoblastic leukemia

We examined myeloid characteristics of myeloid-antigen-positive (My +) and -negative (My −) B-precursor acute lymphoblastic leukemia (ALL) blast cells. Immunophenotyping before and after culture, rearrangements of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes, stimulation of DNA synthesis with granulocyte colony-stimulating factor (G-CSF), and G-CSF binding assay were performed. Of My + ALL blasts, the immunophenotypic staging as B-precursor ALL and rearrangements of IgH and TCR-β, γ and δ genes did not differ from findings in My − ALL blasts. Stimulated with G-CSF, cells from one My + ALL and from one My − ALL patients showed enhancement of DNA synthesis and expression of CD11b and CD13, respectively. G-CSF binding was observed in blasts from 3 My + ALL patients and one My − ALL child. After culture, blasts from My − ALL children expressed CD13 but showed neither enhanced DNA synthesis with G-CSF nor G-CSF binding. Thus, it would appear that (i) My + and My − adult ALL blasts are at the same stage of differentiation; (ii) some My + adult ALL blasts have phenotypic and functional myeloid characteristics; and (iii) induction of CD13 expression in My − ALL after in vitro culture does not correlate with other myeloid characteristics.

Prostaglandin E2 and rat liver regeneration

Background: Prostaglandin (PG) is reported to be involved in hepatic regeneration. However, little is known about the detailed relation in PG-induced stimulation of the proliferation. The present study was attempted to elucidate the relation. Methods: The serial change of PGE2 level released from the regenerating rat liver and the effect of PGE2 on the proliferation of rat hepatocytes were studied, with special reference to PGE2 binding and cyclic AMP (cAMP). Results: The PGE2 level increased 3 hours and 10 hours after partial hepatectomy. Timings of these increases seemed to coincide with those of the first and second increase of cAMP in the liver before the initiation of DNA synthesis. DNA synthesis of hepatocytes in primary culture between 24 and 36 hours, 36 and 48 hours, and 48 and 60 hours of culture were significantly enhanced by addition of PGE2 between 4 and 24 hours of culture at concentrations of 2 nmol/L to 1 μmol/L, 2 nmol/L to 200 nmol/L, and 5 μmol/L to 10 μmol/L, and 2 nmol/L and 10 μmol/L, respectively. Enhancement of DNA synthesis with PGE2 at concentrations less than 1 μmol/L seemed to be associated with the high-affinity binding and that at high concentrations with the lowaffinity binding. Intracellular cAMP level in the hepatocytes increased during culture, and its increase was enhanced by PGE2 addition. Conclusions: It is suggested that PGE2 production in the liver increases biphasically during hepatic regeneration and that PGE2 enhances the proliferation of hepatocytes by a specific receptor-mediated process, which is largely associated with cAMP-dependent process.

Impairment of Myocyte Contractility Following Coronary Artery Narrowing Is Associated with Activation of the Myocyte IGF 1 Autocrine System, Enhanced Expression of Late Growth Related Genes, DNA Synthesis, and Myocyte Nuclear Mitotic Division in Rats

To determine whether the alterations in ventricular loading and myocyte cellular contractile performance produced by short-term coronary artery constriction were associated with the activation of genes implicated in myocyte DNA synthesis including changes in the expression of insulin-like growth factor-1 (IGF 1) and insulin-like growth factor-1 receptors (IGF 1-R), nonocclusive coronary artery narrowing (CAN) was induced in rats. Animals were examined 2 and 7 days after coronary constriction. Following the in vivo documentation of severe impairment of ventricular performance, estimations of single-cell mechanics in vitro showed that peak shortening was decreased in left and right myocytes of coronary stenosed rats. Moreover, time to peak shortening was prolonged whereas velocity of shortening was decreased. These defects in myocyte contractility were accompanied by increases in cell length and width, indicative of myocyte enlargement biventricularly. In addition, CAN led to an enhanced expression of proliferating cell nuclear antigen (PCNA) and histone-H 3 genes in myocytes at 2 and 7 days after surgery. PCNA protein was also detected in these stressed cells. These molecular responses were associated with increases in mRNA for IGF 1 and IGF 1-R in combination with enhanced DNA synthesis and appearance of myocyte nuclear mitotic division. In conclusion, cardiac myocytes may respond to the elevation in wall and myocyte stress by activating an IGF 1-IGF 1-R autocrine system which may modulate the induction of late growth related genes which are essential for DNA replication and myocyte cellular hyperplasia.

Control of Apoptosis and Growth of Malignant T Lymphoma Cells by Lymph Node Stromal Cells

We have previously established the malignant T lymphoma CS-21 cell line from a spontaneous lymphoma in a BALB/c mouse. CS-21 lymphoma cells grew continuously when they were cocultured with stromal CA-12 cells prepared from lymph nodes. CS-21 lymphoma cells, however, could not proliferate by themselves, and they underwent apoptosis when separated from the stromal CA-12 cells. Apoptosis of CS-21 lymphoma cells was determined by observation of morphological changes using a transmission electron microscope and also by detection of nuclear DNA degradative fragments of oligonucleosomal size. Stromal CA-12 cells secreted soluble factors that enhanced DNA synthesis in CS-21 lymphoma cells. The soluble factors, however, were not sufficient to prevent apoptosis of CS-21 cells. Apoptosis of CS-21 lymphoma cells was suppressed only when the CS-21 lymphoma cells were cocultured with substantial numbers of CA-12 cells. The results suggest that the lymph node stromal CA-12 cells played an important role in the growth of CS-21 lymphoma cells by providing at least two different signals. One signal prevented CS-21 cells from apoptotic cell death by direct cell-to-cell contact, and the other signal enhanced the CS-21 cell proliferation by secreted soluble factors.

Adenosine stimulates proliferation of human endothelial cells in culture.

The effect of adenosine on proliferation of human endothelial cells was investigated by adding adenosine to the medium of cultures derived from human umbilical veins. Cell counts on cultures grown in 10 microM adenosine for 4-7 days were 41-53% greater than counts from control cultures. In contrast, 10 microM adenosine had no effect on growth of a human fibroblast cell strain (IMR-90). Neither inosine nor 2',5'-dideoxyadenosine influenced endothelial cell growth at concentrations of 0.1 or 10 microM. Addition of adenosine deaminase abolished the proliferative effect of added adenosine and inhibited proliferation by 16% in control cultures, suggesting that endogenous adenosine may enhance proliferation in culture. The adenosine receptor antagonist, 8-phenyltheophylline, at 0.1 and 1.0 microM blocked the enhanced proliferation caused by 10 microM adenosine. Addition of 10 microM adenosine enhanced DNA synthesis in endothelial cell cultures as indicated by an increased incorporation of [3H]thymidine into acid-insoluble cell material. The results indicate that addition of physiological concentrations of adenosine to human umbilical vein endothelial cell cultures stimulates proliferation, possibly via a surface receptor, and suggest that adenosine may be a factor for human endothelial cell growth and possibly angiogenesis.

Recombinant human insulin-like growth factor-I accelerates recovery and reduces catabolism in rats with ischemic acute renal failure.

This study evaluated whether recombinant human insulin-like growth factor-I (rhIGF-I) enhances recovery of renal function and reduces catabolism in rats with ischemic acute renal failure (ARF). ARF and sham rats received subcutaneous injections of either rhIGF-I or vehicle three times daily starting 5 h after surgery. Serum creatinine and urea, which initially rose similarly in the ARF+vehicle and ARF+rhIGF-I rats, increased more slowly after commencing the rhIGF-I injections. 72 h after surgery, the ARF+rhIGF-I rats, in comparison with ARF+vehicle animals, showed significantly greater renal plasma flow and filtration fraction, a fivefold higher glomerular filtration rate, greater renal cortical IGF-I levels, increased proliferating cell nuclear antigen expression in proximal tubule nuclei and enhanced DNA synthesis in the renal cortex, corticomedullary junction, glomeruli, and tubules as demonstrated by [3H]thymidine incorporation and in corticomedullary junction tubules as determined by autoradiography. Estimated total nitrogen output (ETNO) was greater in ARF+vehicle than in ARF+rhIGF-I or sham rats throughout the study. ETNO in ARF+rhIGF-I rats returned to sham values by the second day after surgery. 72 h after surgery, protein degradation was increased and protein synthesis reduced in the epitrochlearis muscle of ARF+vehicle as compared with ARF+rhIGF-I or sham+vehicle rats. Thus, treatment with rhIGF-I starting 5 h after inducing ischemic ARF in rats increases recovery of renal function, enhances formation of new renal tubular cells, lowers protein degradation, and increases protein synthesis in skeletal muscle and reduces net catabolism.

Stimulation of aged human lung fibroblasts by extracellular ATP via suppression of arachidonate metabolism

The mitogenic effect of extracellular ATP on IMR-90 human fibroblasts subjected to in vitro aging was studied. ATP stimulated DNA synthesis and cell proliferation in young cells as much as epidermal growth factor (EGF) or insulin, while it stimulated aged cells to a much greater extent than seen for any other growth factor tested. When combined with EGF or insulin, ATP restored the greatly reduced mitogenic responsiveness of aged cells nearly to the level noted for young cells. Addition of prostaglandin E2 or other agents that elevate cAMP levels resulted in inhibition of DNA synthesis stimulated by EGF or insulin. Furthermore, the basal release of arachidonic acid and prostaglandin E2 and the endogenous levels of cAMP rose during aging and became much greater than in young cells. All three of these changes were suppressed by extracellular ATP. ATP-dependent suppression of cAMP accumulation was pertussis toxin-sensitive. Protein kinase C down-regulation inhibited arachidonate metabolism and enhanced DNA synthesis stimulated by ATP. These studies suggest that ATP exerts its mitogenic effect, especially on aged IMR-90 cells, at least partially by suppression of arachidonate metabolism.

Foreign body granulomatous inflammation increases the sensitivity of splenocytes to immunomodulation by 1,25-dihydroxyvitamin D 3

1,25-dihydroxyvitamin D 3, the active metabolite of vitamin D, partially inhibits antigen and mitogen-driven lymphocyte stimulation. We studied the effect of granulomatous inflammation on the sensitivity of lymphocytes to 1,25-dihydroxyvitamin D 3 in vitro, measuring the inhibitory effect of 1,25-dihydroxyvitamin D 3 on mitogenesis of splenocytes of mice with chronic inflammation induced by subcutaneous injection of talc. Systematic manifestations of the local inflammation included loss in body weight, splenomegaly, enhanced DNA synthesis by freshly isolated splenocytes and enhanced prostaglandin secretion by activated splenocytes. Splenocytes from animals with local inflammation were more susceptible to inhibition by 1,25-dihydroxyvitamin D 3, but not by prosaglandin E 2. This increased sensitivity to 1,25-dihydroxyvitamin D 3 was abolished by blocking prostaglandin synthesis in splenocyte cultures with indomethacin and was restored by adding prostaglandin E 2. This effect cannot be attributed to enhanced prostaglandin synthesis in the presence of 1,25-dihydroxyvitamin D 3, but is probably due to a qualitative change in the response of splenocytes from inflamed animals to the combined action of 1,25-dihydroxyvitamin D 3 and prostaglandin E 2.

Hepatic stimulator substance protects against acute liver failure induced by carbon tetrachloride poisoning in mice

Hepatic stimulator substance was extracted from the liver of weanling Sprague-Dawley rats according to the method of LaBrecque. Quang-Ming mice were injected with carbon tetrachloride to induce acute liver failure. Hepatic stimulator substance suppressed the elevation of ALT and AST induced by carbon tetrachloride in a dose-dependent manner. Hepatic histological changes indicated that hepatic stimulator substance reduced the severity of hepatic lesion induced by carbon tetrachloride and reversed carbon tetrachloride-induced reduction of hepatic mitochondrial succinic dehydrogenase activity. In attempting to elucidate the mechanism or mechanisms of this protective effect, we found that hepatic stimulator substance significantly restored the carbon tetrachloride-induced decrease of hepatocyte plasmalemma and mitochondrial and microsomal membrane fluidity. Hepatic stimulator substance also decreased the malondialdehyde content of carbon tetrachloride-intoxicated mice; restored the liver-reduced glutathione content, which was lowered by carbon tetrachloride intoxication; stimulated liver regeneration, as shown by enhanced DNA synthesis; and increased the 3H-thymidine incorporation into DNA of hepatocytes. We propose that hepatic stimulator substance protects the liver against acute liver failure induced by carbon tetrachloride poisoning, probably by an antioxidative effect on hepatocyte membrane lipid peroxidation, which was increased by free radicals produced from carbon tetrachloride. In addition, hepatic stimulator substance stimulates hepatocyte proliferation. These protective mechanisms may act in concert to protect against carbon tetrachloride injury.

A role for heparan sulfate in androgen-induced deoxyribonucleic acid synthesis of mouse mammary carcinoma (Shionogi carcinoma 115)-derived SC-3 cells.

In response to androgen stimuli, SC-3 cells cloned from mouse mammary carcinoma (Shionogi carcinoma 115) secrete heparin-binding growth factor, which is able to bind to fibroblast growth factor (FGF) receptor, ultimately resulting in SC-3 cell proliferation. A role for glycosaminoglycan in the process of this androgen-induced autocrine loop was examined. When quiescent SC-3 cells were stimulated with testosterone, enhanced DNA synthesis was demonstrated even at the early phase (12-24 h) of stimulation. During this early period, autocrine growth-promoting activity was found to be associated with cell surface and extracellular matrix, but not to be present in the conditioned medium. This bound form of growth-promoting activity was able to be extracted with 2 M NaCl (pH 7.5) and absorbed onto a heparin-Sepharose affinity column, from which it was eluted at a concentration of 1.1-1.3 M NaCl. Extracted growth factor, whose activity was partially blocked by anti-basic FGF antibody, up-regulated the expression of FGF receptor-1 mRNA. These characteristics were similar to those of a soluble form of SC-3-derived growth factor previously reported from our laboratory. Androgen-induced enhancement of DNA synthesis was inhibited by simultaneous treatment of SC-3 cells with 10 mIU/ml heparitinase or 25 mM sodium chlorate (an inhibitor of phosphoadenosine sulfate synthesis). However, chlorate treatment did not affect the synthesis and distribution of androgen-induced growth factor, suggesting that the response of chlorate-treated cells to this growth factor was impaired. These results indicate that heparan sulfate has important roles in concentrating androgen-induced heparin-binding growth factor on or very close to the cell surface and in potentiating its bioactivity.

Cross‐linking of surface IgM, but not surface IgD receptors, by soluble monoclonal antibodies primes murine B cells to secrete immunoglobulin in response to lymphokines

We have previously reported the development of a two-step culture system in which soluble anti-mu monoclonal antibodies prime small resting murine B cells to secrete immunoglobulin (Ig) in response to restimulation with a mixture of interleukin-4 (IL-4) and IL-5. Here we have extended these studies to investigate the effects of engaging surface IgD (sIgD). We find that, unlike anti-mu, three different anti-delta monoclonal antibodies did not prime B cells to secrete Ig. In addition, these anti-delta antibodies inhibited anti-mu-stimulated priming for Ig secretion, while enhancing DNA synthesis in response to anti-mu. Furthermore, anti-delta antibodies still inhibited anti-mu-induced priming when added 24-48 h after anti-mu. These results therefore suggest that triggering of sIgD on B cells induces a dominant inhibitory signal which is not necessarily dependent upon co-ligation of sIgM and sIgD receptors. In addition, these findings raise the possibility that ligating sIgM or sIgD receptors on mature B cells in the absence of T cell help, may produce different downstream effects.

Effect of elimination diets on food-specific IgE antibodies and lymphocyte proliferative responses to food antigens in atopic dermatitis patients exhibiting sensitivity to food allergens

Peripheral blood mononuclear cells (PBMCs) from patients with atopic dermatitis (AD) selected as being sensitive to hen's egg or cow's milk responded to food antigens, ovalbumin, or bovine serum albumin, with significantly enhanced DNA synthesis compared with the DNA synthesis in PBMCs from nonatopic control subjects and food-sensitive patients with immediate symptoms. Patients were treated with elimination diets. Symptoms of AD had been in remission during elimination diets. The levels of specific IgE antibodies to hen's egg or cow's milk decreased during elimination diets in patients with positive radioallergosorbent test (RAST). In patients with negative RAST, specific IgE antibodies remained negative during elimination diets. The proliferative responses of PBMCs to food antigens also decreased during elimination diets in patients with proliferative responses before elimination diets. Taken together, specific IgE antibodies to food antigens are useful indexes of the effect of elimination diets in food-sensitive patients with AD and positive BAST, and proliferative responses of PBMCs to food antigens are useful indexes of the effect of elimination diets in food-sensitive patients with AD and proliferative responses of PBMCs. (J ALLERGY CLIN IMMUNOL 1993;91:668-79.)

Neurotensin-amplification of DNA synthesis stimulated by EGF or TGF alpha in primary cultures of adult rat hepatocytes.

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. In the presence of neurotensin, the stimulatory effect of epidermal growth factor or transforming growth factor alpha on DNA synthesis was enhanced 3- to 5-fold. Since neurotensin by itself did not stimulate DNA synthesis at all concentrations tested, enhancement of DNA synthesis by neurotensin is due to the amplification of the effect of epidermal growth factor or transforming growth factor alpha. The amplification by neurotensin was observed in a dose-dependent manner with a maximal effect at 10(-8)M, although its effect was significantly observed at as low as 10(-10) M. This neurotensin amplification of DNA synthesis was observed when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that minor components in the medium are required for hepatocytes to fully respond to neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N did not have this amplifying effect on DNA synthesis at any concentration tested. From these results, neurotensin can be regarded as a new secondary mitogen.

<b>POTENTIATION OF HEPATOCYTE SENSITIVITY TO MITOGENS BY GLUTAMIC ACID IN PRIMARY </b><b>CULTURE </b>

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L-15 medium, hepatocyte response to EGF was low compared with those cultured in Williams'medium E or Koga's medium L. Furthermore, in L-15 medium, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes. Addition of glutamic acid (1 to 20 mM) to EGF-containing L-15 medium not only enhanced DNA synthesis over 10-fold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested