Novel mutations of the PRKAR1A gene in patients with acrodysostosis.

Acrodysostosis is characterized by a peripheral dysostosis that is accompanied by short stature, midface hypoplasia, and developmental delay. Recently, it was shown that heterozygous point mutations in the PRKAR1A gene cause acrodysostosis with hormone resistance. By mutational analysis of the PRKAR1A gene we detected four different mutations (p.Arg368Stop, p.Ala213Thr, p.Tyr373Cys, and p.Arg335Cys) in four of seven affected patients with acrodysostosis. The combination of clinical results, endocrinological parameters and in silico mutation analysis gives evidence to suppose a pathogenic effect of each mutation. This assumption is supported by the de novo origin of these mutations. Apart from typical radiological abnormalities of the hand bones, elevated thyroid stimulating hormone and parathyroid hormone values as well as short stature are the most common findings. Less frequent features are characteristic facial dysmorphisms, sensorineural hearing loss and mild intellectual disability. These results lead to the conclusion that mutations of PKRAR1A are the major molecular cause for acrodysostosis with endocrinological abnormalities. In addition, in our cohort of 44 patients affected with brachydactyly type E (BDE) we detected only one sequence variant of PRKAR1A (p.Asp227Asn) with an unclear effect on protein function. Thus, we conclude that PRKAR1A mutations may play no major role in the pathogenesis of BDE.

Intravenous itraconazole followed by oral itraconazole in the treatment of invasive pulmonary aspergillosis in patients with hematologic malignancies, chronic granulomatous disease, or AIDS.

The pharmacokinetics, efficacy, and safety of intravenous (iv) itraconazole (2 days at 400 mg/day, 12 days at 200 mg/day), followed by 12 weeks of oral capsules (400 mg/day) were studied in 31 immunocompromised patients with pulmonary invasive aspergillosis. All patients received iv itraconazole (median duration, 14 days), and 26 then received oral itraconazole (median duration, 78.5 days). After receiving iv itraconazole, concentrations increased rapidly, with trough plasma levels > or =250 ng/mL in 91% of patients and in all patients by day 7. Concentrations > or =500 ng/mL were observed in 64% of patients by day 2. Mean trough concentrations after 2 and 14 days were 670 and 850 ng/mL, respectively. Therapeutic levels were maintained after switching to oral capsules. A complete or partial response was seen at the last on-treatment assessment in 15 (48%) of 31 patients, with 6 (19%) showing stable disease. Itraconazole was well tolerated, with no unexpected effects. Overall iv/oral itraconazole was safe and effective in invasive aspergillosis.

High-performance liquid chromatographic method with electrochemical detection for the analysis of O6-methylguanine.

An improved system consisting of a combination of high-performance liquid chromatographic methods with electrochemical detection for the separation and analysis of the DNA adduct O6-methylguanine (O6MG) has been developed. This adduct is produced by the interaction of methylating agents with DNA and induces mispairing in the DNA of the target cells. A good separation of modified from unmodified bases is first achieved with an HPLC system using a Partisil 10 SCX column and a salt gradient. A second HPLC step with electrochemical detection and a C18 column is used for farther separation and quantitation of O6-methylguanine. This method shows a linear response up to 15 pg of 06MG tested. The lowest amount detected was 0.5 pg of O6MG and is highly reproducible. This method is useful to study DNA damage as a product of cellular metabolism and its effects on the process of carcinogenesis.

Phase I and pharmacokinetic study of the orally administered farnesyl transferase inhibitor R115777 in patients with advanced solid tumors.

R115777 is a novel selective inhibitor of farnesyl transferase, an enzyme that is involved in the proliferation of the malignant cell type. This study was designed to determine the toxicity, maximal tolerated dose and pharmacokinetics of R115777 when given orally b.i.d. for 28 days followed by 1-2 weeks of rest. Patients with advanced solid tumors for whom no standard therapy was available could enter the study. The starting dose of R115777 was 200 mg/dose and inter- as well as intra-patient dose escalations were performed with increments of 100 mg/dose. Nine patients entered the study and received in total 23 treatment cycles. A dose of 300 mg b.i.d. proved feasible with grade 4 neutropenia occurring in one of six patients who completed the first treatment cycle. Other toxicities were infrequent. Pharmacokinetic analysis demonstrated that peak plasma concentrations of 881+/-393 ng/ml were reached within 1-5 h. No accumulation of R115777 was observed over a 28-day period. The study was terminated based on these results together with the observation from a related phase I study in which higher doses of R115777 were associated with the frequent occurrence of grade 3-4 myelosuppression. We conclude that the recommended dose of R115777 given for 28 days followed by 1-2 weeks of rest is 300 mg b.i.d. Myelosuppression is the dose-limiting toxicity.

Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1.

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.

Itraconazole oral solution for primary prophylaxis of fungal infections in patients with hematological malignancy and profound neutropenia: a randomized, double-blind, double-placebo, multicenter trial comparing itraconazole and amphotericin B.

Systemic and superficial fungal infections are a major problem among immunocompromised patients with hematological malignancy. A double-blind, double-placebo, randomized, multicenter trial was performed to compare the efficacy and safety of itraconazole oral solution (2.5 mg/kg of body weight twice a day) with amphotericin B capsules (500 mg orally four times a day) for prophylaxis of systemic and superficial fungal infection. Prophylactic treatment was initiated on the first day of chemotherapy and was continued until the end of the neutropenic period (>0.5 x 10(9) neutrophils/liter) or up to a maximum of 3 days following the end of neutropenia, unless a systemic fungal infection was documented or suspected. The maximum treatment duration was 56 days. In the intent-to-treat population, invasive aspergillosis was noted in 5 (1.8%) of the 281 patients assigned to itraconazole oral solution and in 9 (3.3%) of the 276 patients assigned to oral amphotericin B; of these, 1 and 4 patients died, respectively. Proven systemic fungal infection (including invasive aspergillosis) occurred in 8 patients (2.8%) who received itraconazole, compared with 13 (4.7%) who received oral amphotericin B. Itraconazole significantly reduced the incidence of superficial fungal infections as compared to oral amphotericin B (2 [1%] versus 13 [5%]; P = 0.004). Although the incidences of suspected fungal infection (including fever of unknown origin) were not different between the groups, fewer patients were administered intravenous systemic antifungals (mainly intravenous amphotericin B) in the group receiving itraconazole than in the group receiving oral amphotericin B (114 [41%] versus 132 [48%]; P = 0.066). Adequate plasma itraconazole levels were achieved in about 80% of the patients from 1 week after the start of treatment. In both groups, the trial medication was safe and well tolerated. Prophylactic administration of itraconazole oral solution significantly reduces superficial fungal infection in patients with hematological malignancies and neutropenia. The incidence of proven systemic fungal infections, the number of deaths due to deep fungal infections, and the use of systemic antifungals tended to be lower in the itraconazole-treated group than in the amphotericin B-treated group, without statistical significance. Itraconazole oral solution is a broad-spectrum systemic antifungal agent with prophylactic activity in neutropenic patients, especially for those at high risk of prolonged neutropenia.

Late effects of intraoperative radiation therapy in anastomotic rat colon.

PURPOSE: to determine whether intraoperative radiotherapy causes long-term negative effects on the healing of colonic anastomoses in the rat. METHODS AND MATERIALS: 175 rats were divided into seven equal groups. One group served as sham-irradiated control group. In the others, following a colonic resection, 1 or 2 cm of the distal bowel limb was irradiated with a single dose of 10, 15, or 20 Gy (groups 10/1, 15/1, 20/1, 10/2, 15/2, and 20/2, respectively). Subsequently, an anastomosis was constructed. The animals were killed after 6 (n = 10 in each group) or 12 (n = 15) months. The abdomen was inspected for abnormalities and the colonic diameter was measured. The anastomotic segment was analyzed biochemically (hydroxyproline) and histologically. RESULTS: During the experimental period, 1 rat (group 15/1) died because of anastomotic leakage and 3 others died from unknown causes. There was no difference in colonic diameter between groups. Altogether 17 rats developed an adenocarcinoma in the irradiated area: 11 of these had received a dose of 20 Gy. Histological observation indicated that fibrosis was present only in a limited number of animals, mostly after irradiation with a dose of 15 or 20 Gy. All anastomoses were functional and showed normal histology. The hydroxyproline content of the anastomotic segment was increased--with respect to the control group--only in the 20/2 group after 6 months. After 12 months, the hydroxyproline concentration in the (irradiated) segment distal to the anastomosis proper was higher in the 10/1 and 15/1 groups than in the control group. Otherwise, there were no differences between groups. CONCLUSION: Intraoperative irradiation with a single dose of 10-20 Gy, delivered to the distal limb used for anastomotic construction, does not appear to constitute a threat to anastomotic integrity. Dose-related changes included formation of adenocarcinomas and fibrosis, but function and histology of the anastomosis proper remained unaffected.

Moderate doses of intraoperative radiation severely suppress early strength of anastomoses in the rat colon.

Intraoperative irradiation appears to be a valuable addition to the modalities available to treat patients with large bowel cancer. However, its potential effect on healing of anastomoses has not been investigated extensively. For this purpose, male Wistar rats underwent colonic resection. Subsequently, 1 cm of each bowel end was irradiated with doses of 10, 15, 20 or 25 Gy and intestinal continuity was restored. After 3 or 7 days, animals were killed and the anastomoses were analyzed for bursting pressure (intraluminal force), breaking strength (longitudinal force) and hydroxyproline content. Intraoperative irradiation led to a massive (40-70%) and significant (P < 0.025) reduction in bursting pressure 3 days after operation compared to the control group for every dose used. After 7 days, the bursting site was outside the area of the anastomosis in all groups. The breaking strength at day 3 was also reduced, even after 10 Gy. At day 7, when tearing still occurred in the wound area, the breaking strength was still significantly lower in the 15- and 25-Gy groups than in the control group. The hydroxyproline content of the anastomoses was significantly reduced only after irradiation with the higher doses. Thus intraoperative irradiation constitutes a threat to early strength of anastomoses in the rat colon, and even at moderate doses it may threaten the integrity of the anastomosis.

A semiquantitative histological analysis of repair of anastomoses in the rat colon after combined preoperative irradiation and local hyperthermia.

Hyperthermia is a promising method for increasing the efficacy of radiation therapy of colorectal cancer. To study the histological aspects of healing of an anastomosis in the colon, after combined preoperative (sham) irradiation and (sham) hyperthermia treatment, 48 male Wistar rats were divided randomly into four groups. In each animal, a segment of the colon was treated successively by (sham) irradiation (single dose of 25 Gy X rays) and/or (sham) hyperthermia (44 degrees C, 30 min). After 5 days, a resection of the colon was performed by construction of an anastomosis: The distal limb consisted of (sham-) irradiated and/or (sham-) hyperthermia-treated bowel. Rats were killed 3 or 7 days after the surgical procedure. Evaluation of healing of the anastomosis was made by: (1) histological analysis of sections stained with hematoxylin and eosin, (2) semiquantitative measurement of collagen in the area of the anastomosis and (3) semiquantitative analysis of the number of macrophages by immunocytochemistry. Healing of the anastomoses in animals receiving irradiation or hyperthermia alone and in control animals was relatively uneventful. There were no differences between groups in formation of collagen or infiltration by macrophages in the area of the anastomosis. Animals treated with both radiation and hyperthermia showed marked necrosis, infiltration by polymorphonuclear leukocytes and rupture of the anastomosis. It is concluded that preoperative irradiation with a single dose of 25 Gy in combination with local hyperthermia at 44 degrees C for 30 min leads to disturbed repair of anastomoses.

Intra-operative irradiation prolongs the presence of matrix metalloproteinase activity in large bowel anastomoses of the rat.

There exists a growing interest in intra-operative radiation therapy as a treatment modality for large bowel cancer. In a previous experimental study we showed that high-dose intra-operative irradiation delays the healing of colonic anastomoses. However, the contribution of proteases is unknown. In the present study, the gelatinolytic and collagenolytic activity in the healing anastomoses is investigated. After a resection of a 1-cm length of colon (uninjured colon), the rats were irradiated with a single dose of 25 Gy, either to the proximal limb, referred to as the proximal group, or to both proximal and distal limbs of the bowel, referred to as the combined group, before anastomotic construction. Both groups were compared to a control group with anastomoses which were sham-irradiated. The animals were killed 1, 3 or 7 days after operation. The gelatinolytic activity in uninjured and anastomotic tissue was quantified by gelatin zymography and the collagenolytic activity by an assay using a fibrillar rat collagen substrate. Compared with resected uninjured colon, most of the gelatinolytic activities were markedly increased in anastomotic tissue of all groups during the first postoperative week, and new additional activities were detected. The additional metalloproteinases (the 95-kDa family) of both irradiated groups were significantly elevated compared to the anastomoses of the sham-irradiated control group at 7 days after operation. In anastomotic tissue of all groups, the collagenolytic activity of the tissue was also significantly increased at 1 and 3 days after construction with respect to the resected, uninjured colon. After 7 days this effect had disappeared for the sham-irradiated anastomoses, but the activity in the anastomoses in both the proximal and combined groups remained significantly elevated. The findings provide evidence that intra-operative irradiation prolongs the presence of elevated gelatinolytic and collagenolytic activities in colon anastomoses. It may contribute to a reduced or delayed accumulation of collagen and other matrix proteins that supply anastomotic strength.

Mass-spectrometric analysis of naturally processed peptides recognized by ovarian tumor-associated CD8(+) CTL.

Antigens recognized by cytotoxic T cells (CTL) are expressed as peptides presented by MHC class I molecules. To isolate peptides from the MHC molecule HLA-A2.1 and identify epitopes that define the activity profile of ovarian CD8(+) CTL, peptides were separated by reverse-phase high-pressure liquid chromatography (HPLC), and analyzed by electrospray ionization-tandem mass spectrometry (ES-MS). HLA-A2.1-bound peptides were extracted from the ovarian tumor line SKOV3 transfected with the HLA-A2.1 (clone 1E4) and C1R.A2 cells transfected with HCA-A2.1 and HER-2 (clone HER-2.J) by immunoaffinity chromatography. At least five peaks of distinct retention times (termed 1, 2A, 2B, 2C, and 3) were recognized by an ovarian HER-2(high) (HER-2(hi)) tumor-associated HLA-A2(+), CD8(+) CTL line. ES-MS analysis was performed for peak 2B peptides from both types of cells. In the four consecutive fractions of peak 2B, at least 27 and 16 ion species of mass-to-charge (m/z) ratio between 760-1300 were detected in 1E4 and HER-2.J cells, respectively. The abundance of four 1E4 and six HER-2.J ions believed to be peptides in four consecutive HPLC fractions in this peak matched the CTL activity profile. Of these, two ions with actual m/z ratios 497.3-498.4 and 792.8-793.2, were found in the peak 2B from both types of cells. Since little is known about the tumor Ag recognized in human cancers, characterization of these ions may lead to identification of novel tumor Ag in breast and ovarian cancers. This may also be useful in developing quantitative approaches to the identification of tumor Ag and the determination of epitope density on tumor and normal cells. This may help characterize the relationship between tumor immunity and epitope tolerance in human epithelial cancers.

Reversible inhibition of IL-8 receptor B mRNA expression and proliferation in non-small cell lung cancer by antisense oligonucleotides.

We examined the importance of IL-8 receptor B mRNA expression in the growth of non-small cell lung cancer (NSCLC). Using antisense oligonucleotide ICN 197, we were able to inhibit IL-8 R B mRNA expression in vitro. The sequence specific effect of antisense oligonucleotide and down-regulation of IL-8 R B mRNA was shown by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Southern blot analysis. The proliferation of treated cells was measured by 3H thymidine incorporation. We found that treatment of NSCLC cells caused reversible growth inhibition and reversible down regulation of IL-8 R B mRNA. Furthermore, we observed that the treatment of nude mice with oligonucleotide ICN 197 inhibited the growth of tumors developed from NSCLC cells injected subcutaneously. Our data in vitro suggest that IL-8 receptor B mRNA expression is required to maintain the proliferative rate of NSCLC. Based on the data in vivo. oligonucleotide ICN 197 may be considered for the development of novel therapeutic treatment for lung cancer.

Combined preoperative irradiation and local hyperthermia delays early healing of experimental colonic anastomoses.

OBJECTIVE: To determine if a combination of preoperative irradiation and local hyperthermia of a colonic segment is detrimental to subsequent early anastomotic healing. DESIGN: A prospective randomized experimental trial. SETTING: An animal research laboratory. INTERVENTIONS: Eighty male Wistar rats were randomly divided into 4 groups. In each animal, a segment of the colon was treated successively by (sham) irradiation and (sham) hyperthermia. After 5 days, a colonic resection was performed and an anastomosis was constructed; the distal limb consisted of (sham) irradiated, (sham) hyperthermia-treated bowel. The rats were killed 3 or 7 days after surgery. MAIN OUTCOME MEASURES: Body weight, serum albumin and protein levels, anastomotic bursting pressure, breaking strength, and hydroxyproline content. RESULTS: All animals tolerated (sham) treatment well. Weight was diminished, though not notably, in treated animals vs the control group. After combined preoperative irradiation and hyperthermia, the frequency of local anastomotic complications increased: 4 of 20 animals had a covered perforation when they were killed. In this group, the bursting pressure was lower 3 days after the operation (P = .008). The breaking strength was also lower but not notably. The serum albumin level was significantly lower in this group vs the control group (P = .006); the serum protein level was not decreased. After 7 days, no differences existed between the groups. The hydroxyproline content of the anastomotic tissue was notably higher in rats treated with radiation plus hyperthermia vs control rats (in both the 3- and 7-day groups). The anastomotic hydroxyproline concentration did not differ between the groups. CONCLUSIONS: The combination of preoperative irradiation and hyperthermia results in increased local anastomotic complications. Anastomotic strength is at risk in the first days after the anastomotic reconstruction. Preoperative irradiation or hyperthermia alone does not lead to impaired anastomotic healing in the early phase.

Divergent patterns of matrix metalloproteinase activity during wound healing in ileum and colon of rats.

BACKGROUND: Uncontrolled and increased extracellular matrix degradation during early anastomotic repair in the intestine may reduce wound strength increasing the risk of anastomotic dehiscence. AIMS: To characterise the metalloproteinases present in intact and anastomosed ileum and colon to study their role in matrix degradation after surgery. SUBJECTS: Tissue extracts of uninjured, and of anastomosed rat ileum and colon at postoperative days 1, 2, 3, 7, and 90, were used. METHODS: Metalloproteinases were identified by gelatin and casein zymography. Aminophenylmercuric acetate (APMA) treatment was used to activate latent metalloproteinases. RESULTS: Both uninjured ileum and colon contained a 60 and 67 kDa activity, but a 54 and 72 kDa gelatinase was present in ileum only, and a 51 kDa activity in colon only. APMA treatment converted the 60 kDa protease to 54 and 51 kDa forms and the 72 kDa protease to the 67 kDa form. These gelatinases may correspond to latent and active forms of MMP 1 and MMP 2, respectively. Additional metalloproteinases were observed after anastomotic construction. Both ileum and colon contained 95 and 230 kDa gelatinases, which were converted to 83 and 76 kDa forms by APMA. They may be the latent and active forms of MMP 9, respectively. Gelatinolytic activities of 25 and 28 kDa were only found in anastomosed ileum. Caseinolytic activities were only found in ileum extracts and those were most prominent at day 1, 2, and 3 after surgery. CONCLUSIONS: The metalloproteinase pattern in ileum and colon differ considerably suggesting that matrix degradation after anastomotic construction may also vary.

A tyrphostin-derived inhibitor of protein tyrosine kinases: isolation and characterization.

We recently reported that tyrphostin 23 (3,4-dihydroxybenzylidene malononitrile) is unstable in solution and that some of the degradation products are better inhibitors of the tyrosine kinase activity of Src and the EGF-receptor kinase than the parent compound itself (Ramdas et al., Cancer Res. 54, 867-868, 1994). In this study, the tyrphostin 23-derived compound designated P3, which is a more stable and potent protein tyrosine kinase inhibitor, was isolated. P3 was purified from oxidized tyrphostin 23 by solvent extraction, silica-gel flash chromatography, and reverse-phase high-pressure liquid chromatography. The physical characteristics of the isolated compound were determined and its chemical structure elucidated by 1H and 13C NMR spectroscopy. The proposed structure of this new inhibitor is that of a tyrphostin 23 dimer joined at the benzylidene carbon. P3 was evaluated in vitro as an inhibitor of four different protein tyrosine kinases (Src, Csk, EGF-receptor, and FGF-receptor) and two protein serine kinases (PK-A and PK-C). This compound exhibited the most inhibitory activity against Src with a Ki value of 6 microM and was less inhibitory toward the other protein kinases with Ki values ranging from 35 to 300 microM. P3 did not inhibit other nucleotide-utilizing enzymes such as lactate dehydrogenase and hexokinase. The growth and colony formation of HT-29 colon adenocarcinoma cells that contain activated Src was inhibited by P3 with an IC50 value of approximately 10 microM.

Intraoperative irradiation delays anastomotic repair in rat colon.

BACKGROUND: There exists a growing interest in intraoperative radiation therapy as a treatment modality for large-bowel cancer. Since such therapy could interfere with wound repair, we investigated its effects on early healing of colonic anastomoses. METHODS: After resection of 1 cm of colon, rats were irradiated with a single dose of 25 Gy, either to the proximal limb (P group) or to both proximal and distal limbs of the bowel (PD group) before anastomotic construction. Both groups were compared with a sham-irradiated control group. Animals were killed 3, 7, or 14 days after operation, and healing was assessed by mechanical and biochemical (collagen) parameters. RESULTS: Three days after operation, bursting pressure was significantly lowered in the P group, whereas in the PD group both bursting pressure and breaking strength were strongly reduced. At day 7, the breaking strength was still reduced in the PD group, but not significantly so in the P group. The collagen synthetic capacity of the anastomotic segments was significantly lowered in both irradiated groups at day 3, resulting in a diminished collagen concentration in the actual wound area after 7 days. At 14 days after operation, no differences in strength were found between control and irradiated groups, while anastomotic hydroxyproline levels were significantly higher in both the P and PD groups than in the control group. CONCLUSIONS: High-dose intraoperative radiation therapy delays the healing of colonic anastomoses; it transiently reduces strength, probably as a result of a diminished accumulation of collagen.

Glutamate induces the growth factors NGF, bFGF, the receptor FGF-R1 and c-fos mRNA expression in rat astrocyte culture.

The effect of glutamate on primary cultures of rat cortical astrocytes was studied using Northern blot hybridization. Incubation with glutamate (100 microM, 15 min) induced nerve growth factor (NGF), basic fibroblast growth factor (bFGF), FGF receptor (FGF-R1) and proto-oncogene c-fos gene expression in a time dependent manner. Maximal induction of NGF, bFGF and FGF-R1 mRNA was reached after 4 h of incubation (7.2-fold induction of NGF, 3-fold increase in bFGF and 3.6-fold induction of FGF-R1 mRNA). The induction kinetics of NGF, bFGF and FGF-R1 mRNA are similar. The rapid (1 h) 77-fold induction of the c-fos transcript precedes the induction of the other genes tested.

Opposite functions of jun-B and c-jun in growth regulation and neuronal differentiation.

Induction of the jun-B and/or c-jun transcription factors is part of the immediate early response to diverse stimuli that induce alterations in cellular programs. While c-jun is a protooncogene whose expression is required for induction of cell proliferation, jun-B has recently been found to be induced by stimuli inducing differentiation in various cell lines. Furthermore, its expression is largely restricted to differentiating cells during embryogenesis. To determine the functional significance of these findings, we used antisense phosphorothioate oligodeoxynucleotides to inhibit expression of the two genes in proliferating and neuronally differentiating cells. While selective inhibition of c-jun expression reduced proliferation rates, inhibition of jun-B protein synthesis markedly increased proliferation in 3T3 fibroblasts, human mammary carcinoma cells and PC-12 pheochromocytoma cells, suggesting jun-B involvement in negative growth control. Neuronal differentiation of PC-12 cells induced by nerve growth factor (NGF) was prevented by inhibition of jun-B protein synthesis. PC-12 cells not only failed to grow neurites but also remained in the proliferative state. Furthermore, in cultured primary neurons from rat hippocampus, inhibition of jun-B expression, again, markedly reduced morphological differentiation. Conversely, inhibition of c-jun protein synthesis enhanced morphological differentiation of both primary neurons and PC-12 tumor cells. Thus, jun-B expression is required for neuronal differentiation and its balance with c-jun activity is involved in regulating key steps in proliferation and differentiation processes.