DNA hypermethylation of extracellular matrix-related genes in human periodontal fibroblasts induced by stimulation for a prolonged period with lipopolysaccharide derived from Porphyromonas gingivalis.

OBJECTIVES AND BACKGROUND: The involvement of DNA methylation in periodontal disease is not clear. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis is involved in the progression of periodontal disease. We recently developed an in vitro model of LPS infection in human periodontal fibroblast cells (HPdLFs) for a prolonged period. In this study, we examined genome-wide analysis of DNA methylation in HPdLFs stimulated with LPS derived from P. gingivalis for a prolonged period. We noted the hypermethylation of extracellular matrix (ECM)-related genes and examined whether hypermethylation affected their transcription levels. MATERIAL AND METHODS: HPdLFs were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The culture was repeated, alternating 3 d with LPS derived from P. gingivalis and 3 d without LPS for 1 mo. Untreated samples were used as controls. DNA was analyzed using the human CpG island microarray. Quantitative methylation-specific polymerase chain reaction was carried out to confirm reproducibility of the microarray data. The expression levels of mRNA of the selected ECM-related genes from the data were analyzed by quantitative reverse transcription-polymerase chain reaction. RESULTS: We found 25 ECM-related genes with hypermethylation at the CpG island of the promoter region, which exhibited a fourfold greater hypermethylation than controls. Among these genes, hypermethylation of nine ECM-related genes, FANK1, COL4A1-A2, 12A1 and 15A1, LAMA5 and B1, MMP25, POMT1 and EMILIN3, induced a significantly downregulated expression of their mRNA. CONCLUSION: These results indicate that LPS derived from P. gingivalis may cause DNA hypermethylation of some ECM-related genes followed by downregulated expression of their transcriptional levels.

Study Protocol of a Comprehensive Activity Promotion Program for the Prevention of Dementia: A Randomized Controlled Trial Protocol.

BACKGROUND: Several technical devices are available to monitor and promote changes in behavior toward higher activity. In particular, smartphones are becoming the primary platform for recognizing human activity. However, the effects of behavior change techniques that promote physical, cognitive, and social activities on incident dementia in older adults remain unknown. OBJECTIVES: This randomized controlled trial aims to examine the effects of behavior change techniques on the prevention of dementia among community-dwelling older adults using a smartphone as a behavior change tool. DESIGN: A randomized controlled trial. SETTING: Community in Japan. PARTICIPANTS: The study cohort comprises 3,498 individuals, aged ≥60 years, randomized into two groups: the smartphone group (n = 1,749) and the control group (n = 1,749). INTERVENTION: The smartphone group will be asked to use smartphone applications for at least 30 minutes daily to self-manage and improve their physical, cognitive, and social activities. The smartphone group will perform 60-minute group walking sessions using application-linked Nordic walking poles with cognitive stimulation twice a week during the intervention period. The walking poles are a dual-task exercise tool that works with a smartphone to perform cognitive tasks while walking, and the poles are equipped with switches to answer questions for simple calculation and memory tasks. The smartphone and control groups will receive lectures about general health that will be provided during the baseline and follow-up assessments. MEASUREMENTS: Incident dementia will be detected using cognitive tests (at baseline, after 15 months, and after 30 months) and by preparing diagnostic monthly reports based on data from the Japanese Health Insurance System. Participants without dementia at baseline who will be diagnosed with dementia over the 30-month follow-up period will be considered to have incident dementia. CONCLUSIONS: This study has the potential to provide the first evidence of the effectiveness of information communication technology and Internet of Things in incident dementia. If our trial results show a delayed dementia onset for self-determination interventions, the study protocol will provide a cost-effective and safe method for maintaining healthy cognitive aging.

Association of Daily Physical Activity with Disability in Community-Dwelling Older Adults With/Without Chronic Kidney Disease.

OBJECTIVES: Physical activity is recommended for disability prevention in the older adult population; however, the level of physical activity required for older adults with chronic kidney disease (CKD) remains unknown. This study aimed to examine the associations between daily physical activity and disability incidence in older adults with and without CKD to determine relevant daily physical activity levels. DESIGN: Prospective observational study. SETTING AND PARTICIPANTS: 3,786 community-dwelling older adults aged ≥65 years. MEASUREMENTS: Mean daily times spent in light- (LPA) and moderate-to-vigorous physical activity (MVPA) were measured using triaxial accelerometers. CKD was defined by a creatinine estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2. Disability incidence was identified as long-term care insurance certification during a 60-month follow-up period. Associations between physical activity and disability incidence were examined using Cox proportional hazard models stratified by the CKD status. Non-linear and linear associations were tested using the restricted cubic spline. RESULTS: A total of 1,054 individuals were identified to have CKD. Disability incidence was higher in the CKD group than in the non-CKD group. The adjusted cox proportional hazard models indicated that a 10-minute increase in MVPA time was associated with lower disability incidence in the non-CKD group (hazard ratio [HR], 0.838; 95% confidence interval [CI]: 0.764-0.918) and the CKD group (HR, 0.859; 95% CI: 0.766-0.960). Linear associations were observed in MVPA for the non-CKD and CKD groups. CONCLUSION: Increasing MVPA was associated with lower disability incidence in older adults with and without CKD. These findings can help devise disability prevention strategies for older CKD patients.

Association between Non-Face-to-Face Interactions and Incident Disability in Older Adults.

OBJECTIVES: This observational prospective cohort study, conducted between September 2015 and February 2019, aimed to investigate the association between the incidence of disability and non-face-to-face interactions among community-dwelling older adults in Japan. DESIGN: Participants reported their interaction status using a self-report questionnaire. Face-to-face interactions comprised in-person meetings, while virtual interactions (e.g., via phone calls or emails) were defined as non-face-to-face interactions. We examined the relationship between their interaction status at baseline and the risk of disability incidence at follow-up. We also considered several potential confounding variables, such as demographic characteristics. SETTING: The National Center for Geriatrics and Gerontology-Study of Geriatric Syndromes. PARTICIPANTS: We included 1159 adults from Takahama City aged ≥75 years (mean age ± standard deviation = 79.5 ± 3.6 years). MEASUREMENTS: Interaction status was assessed using a self-reported questionnaire consisting of two sections (face-to-face and non-face-to-face interactions), and four questionnaire items. Based on the responses we categorized study participants into four groups: "both interactions," "face-to-face only," "non-face-to-face only," and "no interactions." RESULTS: Individuals with both kinds of interactions (49.3/1000 person-years) or only one kind of interaction (face-to-face = 57.7/1000 person-years; non-face-to-face = 41.2 person-years) had lower incidence of disability than those with no interactions (88.9/1000 person-years). Moreover, the hazard ratios adjusted for potential confounding factors for the incidence of disability in the both interaction, face-to-face-only, and non-face-to-face only groups were 0.57 (confidence interval = 0.39-0.82; p = 0.003), 0.66 (confidence interval = 0.44-0.98; p = 0.038), and 0.47 (confidence interval = 0.22-0.99; p = 0.048), respectively. CONCLUSION: Considering the interaction status of older adults in their day-to-day practice, clinicians may be able to achieve better outcomes in the primary prevention of disease by encouraging older adults to engage in any form of interaction, including non-face-to-face interactions.

Application of self-efficacy theory in dental clinical practice.

In clinical practice, self-efficacy refers to how certain a patient feels about his or her ability to take the necessary action to improve the indicators and maintenance of health. It is assumed that the prognosis for patient behaviour can be improved by assessing the proficiency of their self-efficacy through providing psychoeducational instructions adapted for individual patients, and promoting behavioural change for self-care. Therefore, accurate assessment of self-efficacy is an important key in daily clinical preventive care. The previous research showed that the self-efficacy scale scores predicted patient behaviour in periodontal patients and mother's behaviour in paediatric dental practice. Self-efficacy belief is constructed from four principal sources of information: enactive mastery experience, vicarious experience, verbal persuasion, and physiological and affective states. Thus, self-efficacy can be enhanced by the intervention exploiting these sources. The previous studies revealed that behavioural interventions to enhance self-efficacy improved oral-care behaviour of patients. Therefore, assessment and enhancement of oral-care specific self-efficacy is important to promote behaviour modification in clinical dental practice. However, more researches are needed to evaluate the suitability of the intervention method.

Visceral Fat Accumulation Is Associated with Mild Cognitive Impairment in Community-Dwelling Older Japanese Women.

OBJECTIVES: Visceral fat accumulation is detrimental for brain health and is associated with cognitive impairment in older adults. The objectives of the present study were to examine the association between visceral fat accumulation and prevalence of mild cognitive impairment and its subtypes. DESIGN: a cross-sectional study. PARTICIPANTS: This study enrolled 6,109 community-dwelling older adults, including 3,434 women (mean age: 74.4 years) and 2,675 men (mean age: 74.3 years). Individuals with dementia, Parkinson's disease, stroke, Mini-Mental State Examination scores ≤23, and who could not perform basic activities of daily living independently were excluded. MEASUREMENTS: Participants underwent neurocognitive assessments to assess mild cognitive impairment (MCI) and its subtypes. Visceral fat area (VFA) was measured using abdominal bioelectrical impedance analysis. Participants were divided into quartile groups by VFA. RESULTS: There were 731 (21.3%) women and 562 (21.0%) men with MCI, and the median VFA values were 63.3 cm2 and 96.3 cm2, respectively. Women participants in the second (adjusted odds ratios [aOR], 0.71; 95% confidence interval [95% CI], 0.54-0.94), third (aOR, 0.66; 95% CI, 0.47-0.92), and fourth quartiles of VFA (aOR, 0.62; 95% CI, 0.41-0.93) had a significantly lower risk of MCI than those in the first quartile. Higher VFA quartiles in women were associated with lower risk of non-amnestic MCI. There were no significant differences in men between quartiles. CONCLUSIONS: Visceral fat accumulation was associated with MCI, especially non-amnestic MCI, in community-dwelling older Japanese women. These results suggest that visceral fat accumulation is partially protective against cognitive impairment.

p53: a frequent target for genetic abnormalities in lung cancer.

Allele loss is a hallmark of chromosome regions harboring recessive oncogenes. Lung cancer frequently demonstrates loss of heterozygosity on 17p. Recent evidence suggests that the p53 gene located on 17p13 has many features of such an antioncogene. The p53 gene was frequently mutated or inactivated in all types of human lung cancer. The genetic abnormalities of p53 include gross changes such as homozygous deletions and abnormally sized messenger RNAs along with a variety of point or small mutations, which map to the p53 open reading frame and change amino acid sequence in a region highly conserved between mouse and man. In addition, very low or absent expression of p53 messenger RNA in lung cancer cell lines compared to normal lung was seen. These findings, coupled with the previous demonstration of 17p allele loss in lung cancer, strongly implicate p53 as an anti-oncogene whose disruption is involved in the pathogenesis of human lung cancer.

Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer.

The p53 gene initially was thought to be an oncogene, but recent evidence suggests that wild-type p53 can function as a tumor suppressor gene in lung, colon, and breast cancer as well as less common malignancies. This study reports the first identification of intronic point mutations as a mechanism for inactivation of the p53 tumor suppressor gene. Abnormally sized p53 mRNAs found in a small cell and a non-small cell lung cancer cell line were characterized by sequence analysis of cDNA/PCR products, the RNase protection assay and immunoprecipitation. These mRNAs were found to represent aberrant splicing leading to the production of abnormal or no p53 protein. Sequence analysis of genomic DNA revealed that a point mutation at the splice acceptor site in the third intron or the splice donor site in the seventh intron accounts for the abnormal mRNA splicing. In one patient the same intronic point mutation was found in the tumor cell line derived from a bone marrow metastasis and in multiple liver metastases but not in normal DNA, indicating that it occurred as a somatic event before the development of these metastases. These findings further support the role of inactivation of the p53 gene in the pathogenesis of lung cancer and indicate the role of intronic point mutation in this process.

Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic leukemia.

The p53 gene was examined in primary lymphoblasts of 25 pediatric patients with acute lymphoblastic leukemia by the RNase protection assay and by single strand conformation polymorphism analysis in 23 of 25 cases. p53 mutations were found to occur, but at a low frequency (4 of 25). While all four mutations were identified by single strand conformation polymorphism, the comparative sensitivity of RNase protection was 50% (2 of 4). Heterozygosity was retained at mutated codons in 3 of 4 cases. One pedigree was consistent with the Li-Fraumeni syndrome, and bone marrow from both diagnosis and remission indicated a germline G to T transversion at codon 272 (valine to leucine). Although members of another family were affected with leukemia, a 2-bp deletion in exon 6 was nonhereditary. The other two nonhereditary p53 mutations included a T to G transversion at codon 270 (phenylalanine to cysteine) and a G to C transversion at codon 248 (arginine to proline). These data support the role of both hereditary and acquired p53 mutations in the pathogenesis and/or progression of some cases of childhood acute lymphoblastic leukemia.

Occurrence of p53 gene abnormalities in gastric carcinoma tumors and cell lines.

We explored the state of the p53 gene in gastric cancer. Using one or more methods, we examined 15 specimens from primary carcinomas (14 tumors, one cell line), five cell lines derived from metastases, and seven paired samples of nonmalignant gastric mucosa. Sequence analyses of complementary DNA containing the entire p53 gene open reading frame demonstrated abnormalities in one of five samples from primary tumors and in all five samples from metastases. The single cell line derived from a primary carcinoma had no abnormality of the gene. The six abnormalities included four point mutations, one base-pair deletion resulting in a frame shift, and a 24 base-pair deletion caused by an intronic point mutation (as determined by sequence analysis of genomic DNA). Four of the six mutations mapped to regions highly conserved among species or involved in simian virus 40 T-antigen binding. Restriction fragment length polymorphism studies confirmed that chromosome 17p allelic deletions occur only in a minority of primary tumors, but that they may occur more frequently in metastases. Northern blotting and ribonuclease protection assays detected only a fraction of the p53 gene abnormalities detected by sequencing. Our findings indicate that mutations of the p53 gene are relatively rare in primary gastric tumors but appear to be relatively frequent in cell lines derived from metastatic lesions. Our results may help in understanding the molecular events associated with progression and metastasis in gastric carcinoma.

Growth-associated shedding of a tumor antigen (CE7) detected by a monoclonal antibody.

A monoclonal antibody was developed against an antigen, termed CE7, which was highly expressed on the surface of rat fibrosarcoma KMT-17 cells (clone A3) cultured in low serum medium (A3-1% FCS). The CE7 antigen was not detectable on A3 cells cultured in ordinary high serum medium (A3-10% FCS), on in vivo passaged A3 cells, or on parental in vivo KMT-17 cell line. However, immunoelectron microscopy and Western blot analyses indicated that CE7 antigen was produced by these tumor cells in all circumstances but was shed from their surfaces in vesicular form into the surrounding tissue culture medium or ascites, unless low serum concentration prevailed and disappeared from their cell surfaces. We have previously reported that the immunogenicity of A3 cells was increased when the serum concentration was lowered from 10% to 1% and the phenomenon paralleled the CE7 antigen expression on the A3 cells. These results suggest that the CE7 antigen could be a tumor-associated rejection antigen and that the expression of the CE7 antigen on A3-1% FCS cells (which is shed by high serum culture or in vivo transplantation and disappears from the cell surface) may play a role in immunological responses against the tumor cells.

Enhanced immunogenicity of the cultured rat fibrosarcoma KMT-17 by cultivation in a low concentration of fetal calf serum.

We examined in different culture conditions alterations in the tumorigenicity and immunogenicity of an A3 clone that had been derived from a rat fibrosarcoma KMT-17. When a fetal calf serum concentration in a culture medium was lowered from 10 to 1%, the tumorigenicity was diminished while the immunogenicity was enhanced in a reversible manner; this was accompanied by a reversible prolongation of the in vitro doubling time. These phenomena were not due to an increase in the quantities of the original tumor-associated antigen and/or of the rat major histocompatibility complex (RT1) but seemed to be due to the appearance of a unique antigen(s) that was detected by an antibody taken from rats immunized with A3 tumor cells cultured in the low fetal calf serum concentration; this antigen(s) may consist of glycoprotein and exist as a crypt antigen(s). These phenomena were measured by an absorption test and flow cytometric analysis. Our observations suggest that the in vitro culture condition of tumor cells, in particular their culturing in the low fetal calf serum concentration medium, modifies the surface of tumor cells and causes a diminishment in their tumorigenicity and an enhancement of their immunogenicity.

Mutations in the p53 gene in primary human breast cancers.

Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.

Polymorphism at codon 213 within the p53 gene.

This report describes a rare polymorphism at codon 213 (silent alteration of CGA to CGG) within the coding region of the p53 gene. The rare polymorphic allele was present in six cases out of 189 lung and breast cancer DNAs analyzed (3.2%) and resulted in the loss of a TaqI site. This allele could be mistaken for a mutation when screening methods of mutation analysis are used without comparison with normal tissue DNA.

Temperature-sensitive mutants of p53 associated with human carcinoma of the lung.

We have compared the effects of specific point mutations on the tertiary and quaternary structure of the human p53 protein. Eight mutants, each derived from primary resected tissues of lung carcinomas, were expressed in vitro under strictly defined conditions, such that the only known variant was the point mutation present in each p53 mRNA. All the mutations were located in highly conserved domains. The tertiary structure of each mutant protein was investigated by reactivity with anti-p53 monoclonal antibodies directed against conformation-dependent epitopes. Quaternary structure was examined by gel filtration. Although all the mutant proteins exhibited abnormal tertiary structures, their quaternary structures appeared similar to wild type, the one exception being p53-tyr135, which contains tyrosine in place of cysteine at residue 135. The conformational phenotype of mutant human p53 was found to be dependent upon (i) the locus of the mutation and (ii) the nature of the amino acid substitution: two different substitutions at residue 273 yielded two mutants with differing structural properties. We have discovered three mutants of human p53 that are temperature sensitive for conformation; one is mutated at codon 273, a 'hotspot' for p53 mutation in human cancer.

Multiple oral squamous epithelial lesions: are they genetically related?

The development of second primary tumors (SPTs) in patients with head and neck squamous cell carcinoma (HNSCC) has become an increasingly important factor in clinical treatment decisions. Currently, clinical and histologic parameters are used to determine whether or not SPT is present. Recent studies suggest that many SPTs in the upper aerodigestive tract have a common clonal origin, challenging the longstanding multiclonal origin concept. To determine genetic relationships among multiple oral cancerous and precancerous lesions (MOCP), we analysed 100 lesions from 26 Japanese patients. Lesion development was synchronous and metachronous. We looked for patterns of microsatellite alterations (MA) using seven markers at chromosomes 3p14, 9p21, and 17p13, where MA occurs early in oral carcinogenesis. Loss of heterozygosity (LOH) was found in 52.6% (41/78), 62.5% (60/96), and 59.3% (32/54) of informative MOCP at 3p14, 9p21, and 17p13, respectively. Microsatellite instability (MI) was observed in 11, 26 and 13% of the samples at 3p14, 9p21, and 17p13 markers, respectively. Patterns of MA were concordant in only nine (14%) of 63 lesions from four (18%) of 22 patients who initially presented with noninvasive lesions. However, two of four patients with invasive cancer as indexed lesion showed 16 (43%) clonally related MOCP among 37 lesions (P=0.003). The results suggest that the majority of MOCP arise from clonally independent cells affected by field cancerization. However, the probability of mucosal spread of clonal malignant or premalignant cells may increase along with malignant progression.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay.

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT-PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR-SSCP) of exon 5-8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR-SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

Mutations in the p53 gene and human papillomavirus infection as significant prognostic factors in squamous cell carcinomas of the oral cavity.

The p53 gene has been indicated to be a tumour suppressor gene that is found in mutated form in common human cancers. Human papillomavirus (HPV) has oncogenic activity in cervical and oral squamous cell carcinomas (SCCs). The E6 protein of HPV is known to bind with p53 protein and inactive the tumor suppressor activity by promoting p53 degradation. Because of this background, we examined 38 primary, resected specimens of oral SCCs for detection of p53 mutations and HPV DNAs. Exons 5 through 8 of the p53 Mutations were observed in nine cases (24%). HPV-DNA detection and typing were performed using PCR with ¿high risk group' HPV-specified primers. HPV DNA sequences were detected in eight cases (21%). The AvaII digestion pattern of PCR-amplified HPV DNA showed that HPV-16 was present in all eight cases. Seven cases were p53 mutation-positive/HPV-negative, six cases were p53 mutation-negative/HPV-positive, and two intraosseus SCC cases were p53 mutation-positive/ HPV-positive. Thus, 15/38 (40%) cases had inactivation of the p53 protein. Interestingly, p53 mutation-negative/ HPV-negative cases had a poorer prognosis than p53 mutation positive or HPV-positive cases (P < 0.01). We conclude that (1) mutation in the p53 gene and/or HPV infection are frequent (40%) in oral SCC; (2) inactivation of p53 function by mutation and HPV infection are important genetic events in the development of 40% integral of oral SCCs; (3) p53 mutation and HPV infection are not mutually exclusive events and (4) other oncogenes or tumor suppressor genes may be crucial in the development of oral SCC if the prognosis is poor.

Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Lung Cancer Study Group.

The p53 gene has been implicated as a tumor suppressor gene with mutations found in common human cancers. We examined 51 early stage, primary, resected non-small cell lung cancer specimens using an RNAase protection assay and cDNA sequencing. Mutations changing the p53 coding sequence were found in 23/51 (45%) tumor specimens, but not in the corresponding normal lung, were distributed between codons 132 to 283, and included tumors with and without 17p allele loss. Fifteen of the 23 mutations lay in the predicted binding regions for SV40 large T antigen, and 14 were located in regions highly conserved between species. G to T transversions were a common result of p53 mutations in lung cancer compared to other cancers suggesting exposure to different mutagens. In univariate and multivariate analysis the presence of p53 mutations was associated with younger age and squamous histology. However, the presence of p53 mutations was not significantly associated with tumor stage, nodal status or sex and was found in all histologic types of lung cancer. We conclude that somatic mutations in the p53 gene play an important role in the pathogenesis of early stage non-small cell lung cancer.

Unresponsiveness of both non-rearranged and rearranged c-myc to serum stimulation in a mouse plasmacytoma S194.

Expression of non-rearranged and rearranged c-myc in a mouse plasmacytoma cell line S194 cultured in different serum concentration or temperature was examined. Exponentially growing S194 cells expressed a high level of rearranged c-myc and a low level of non-rearranged c-myc mRNAs. The levels of the two c-myc mRNAs were nearly the same in the cells in which growth was suppressed by lowering serum concentrations or incubation temperature in culture medium. Furthermore, even when serum-deprived S194 cells resumed growth following serum restimulation, the induction of non-rearranged c-myc mRNA, observed in a mouse T-cell lymphoma cell line BW5147, was not demonstrated. In contrast to the unchanged c-myc expression, the level of N-ras mRNA was related to changes in cell growth rate induced by changes in serum concentration in S194 cells. These results suggest that not only the rearranged c-myc but also the non-rearranged c-myc is unresponsive to serum stimulation and temperature changes, even though cell growth rate is markedly changed.