Characterization of dental pulp stem cells isolated from a patient diagnosed with Crouzon syndrome.

Stem cells isolated from patients with rare diseases are important to elucidate their pathogeny and mechanisms to enable regenerative therapy. However, the mechanisms underlying tissue regeneration using patient-derived dental pulp stem cells (DPSCs) are unclear. In this study, we investigated the levels of mRNA and protein expression related to cellular differentiation of Crouzon syndrome patient-derived DPSCs (CS-DPSCs) with a Gly338Arg fibroblast growth factor receptor 2 mutation. Multipotency-related gene expression levels were equivalent in both healthy donor DPSCs and CS-DPSCs. CS-DPSCs showed higher osteocalcin (OCN) expression than healthy donor DPSCs. CS-DPSCs showed a lower increase in the rate of OCN expression among phorbol 12-myristate 13-acetate (PMA)-treated cells than healthy donor DPSCs compared with untreated control cells. CS-DPSCs showed a lower phosphorylation rate of p38 and p44/42 in PMA-treated cells than healthy donor DPSCs compared with untreated control cells. These results demonstrate that CS-DPSCs have higher OCN expression and lower PMA stimulation-responsiveness than healthy donor DPSCs.

Characterization of proliferation, differentiation potential, and gene expression among clonal cultures of human dental pulp cells.

Mesenchymal stem cells are a highly promising source of cells for regeneration therapy because of their multilineage differentiation potential. However, distinct markers for mesenchymal stem cells are not well-established. To identify new candidate marker genes for multipotent human dental pulp stem cells, we analyzed the characteristics and gene expression profiles of cell clones obtained from a single dental pulp specimen derived from an 11-year-old female patient. Fifty colony-forming single cell-derived clones were separately cultured until the cessation of growth. These clones varied in their proliferation abilities and surface marker (STRO-1 and CD146) expression patterns, as well as their odontogenic, adipogenic, and chondrogenic differentiation potentials. Four clones maintained their original differentiation potentials during long-term culture. Gene expression profile by DNA microarray analysis of five representative clones identified 1227 genes that were related to multipotency. Ninety of these 1227 genes overlapped with genes reportedly involved in 'stemness or differentiation'. Based on the predicted locations of expressed protein products and large changes in expression levels, 14 of the 90 genes were selected as candidate dental pulp stem cell markers, particularly in relation to their multipotency characteristics. This characterization of cell clones obtained from a single specimen of human dental pulp provided information regarding new candidate marker genes for multipotent dental pulp stem cells, which could facilitate efficient analysis or enrichment of multipotent stem cells.

Dental Pulp Stem Cells: Advances to Applications.

Dental pulp stem cells (DPSCs) have a high capacity for differentiation and the ability to regenerate a dentin/pulp-like complex. Numerous studies have provided evidence of DPSCs' differentiation capacity, such as in neurogenesis, adipogenesis, osteogenesis, chondrogenesis, angiogenesis, and dentinogenesis. The molecular mechanisms and functions of DPSCs' differentiation process are affected by growth factors and scaffolds. For example, growth factors such as basic fibroblast growth factor (bFGF), transforming growth factor-ß (TGF-ß), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and bone morphogenic proteins (BMPs) influence DPSC fate, including in differentiation, cell proliferation, and wound healing. In addition, several types of scaffolds, such as collagen, hydrogel, decellularized bioscaffold, and nanofibrous spongy microspheres, have been used to characterize DPSC cellular attachment, migration, proliferation, differentiation, and functions. An appropriate combination of growth factors and scaffolds can enhance the differentiation capacity of DPSCs, in terms of optimizing not only dental-related expression but also dental pulp morphology. For a cell-based clinical approach, focus has been placed on the tissue engineering triad [cells/bioactive molecules (growth factors)/scaffolds] to characterize DPSCs. It is clear that a deep understanding of the mechanisms of stem cells, including their aging, self-renewal, microenvironmental homeostasis, and differentiation correlated with cell activity, the energy for which is provided from mitochondria, should provide new approaches for DPSC research and therapeutics. Mitochondrial functions and dynamics are related to the direction of stem cell differentiation, including glycolysis, oxidative phosphorylation, mitochondrial metabolism, mitochondrial transcription factor A (TFAM), mitochondrial elongation, and mitochondrial fusion and fission proteins. This review summarizes the effects of major growth factors and scaffolds for regenerating dentin/pulp-like complexes, as well as elucidating mitochondrial properties of DPSCs for the development of advanced applications research.

CD146 positive human dental pulp stem cells promote regeneration of dentin/pulp-like structures.

CD146 and STRO-1 are endothelial biomarkers that are co-expressed on the cellular membranes of blood vessels within human dental pulp tissue. This study characterized the percentage of dentin-like structures produced by CD146-positive (CD146+) human dental pulp stem cells (DPSCs), compared with their CD146-negative (CD146-) counterparts. DPSC populations were enriched using magnetic-activated cell sorting (MACS), yielding CD146+ and CD146- cells, as well as mixtures composed of 25% CD146+ cells and 75% CD146- cells (CD146+/-). Cell growth assays indicated that CD146+ cells exhibit an approximate 3-4 h difference in doubling time, compared with CD146- cells. Cell cycle distributions were determined by flow cytometry analysis. The low percentage of CD146+ cells' DNA content in G0/G1 phase were compared with CD146- and non-separated cells. In contrast to CD146- and non-separated cells, prompt mineralization was observed in CD146+ cells. Subsequently, qRT-PCR revealed high mRNA expression of CD146 and Alkaline phosphatase in mineralization-induced CD146+ cells. CD146+ cells were also observed high adipogenic ability by Oil red O staining. Histological examinations revealed an increased area of dentin/pulp-like structures in transplanted CD146+ cells, compared with CD146- and CD146+/- cells. Immunohistochemical studies detected dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP), as well as human mitochondria, in transplanted DPSCs. Co-expression of CD146 and GFP indicated that CD146 was expressed in transplanted CD146+ cells. CD146+ cells may promote mineralization and generate dentin/pulp-like structures, suggesting a role in self-renewal of stem cells and dental pulp regenerative therapy.

In vivo stem cell transplantation using reduced cell numbers.

Dental pulp stem cell (DPSC) characterization is essential for regeneration of a dentin/pulp like complex in vivo. This is especially important for identifying the potential of DPSCs to function as stem cells. Previously reported DPSC transplantation methods have used with huge numbers of cells, along with hydroxyapatite/tricalcium phosphate (HA/TCP), gelatin and fibrin, and collagen scaffolds. This protocol describe a transplantation protocol that uses fewer cells and a temperature-responsive cell culture dish.

Expression status of mRNA for sex hormone receptors in human dental pulp cells and the response to sex hormones in the cells.

OBJECTIVES: Sex hormone receptors are reported to be present in human dental pulp (HDP) cells. The purpose of this study was to examine the biological significance of oestrogen and androgen receptors (ER and AR, respectively) in HDP cells. DESIGN: We isolated HDP cells expressing ER- and AR-mRNAs and investigated the expression status of the receptors and the response to sex hormones in the cells. RESULTS: HDP cells expressing ER- and/or AR-mRNAs had the ability to form alizarin red S-positive nodules in which calcium and phosphorus were deposited in vitro and to differentiate into odontoblasts-like cells and dentine-like tissue in vivo. Individual clones isolated from HDP cells exhibited a different expression pattern of mRNA for ER and AR. Some clones expressed ERα- and/or ERß-mRNAs and the others coexpressed ER- and AR-mRNAs. Using the Ingenuity software, we found that 17ß-estradiol (E2) and dihydrotestosterone (DHT) could act directly on HDP cells through ER-or androgen signalling-mediated mechanisms. E2 or DHT stimulated the mRNA expression for genes related to odontogenesis of dentine-containing teeth and odontoblast differentiation, suggesting that ER and AR in HDP cells may be involved in dentinogenesis. CONCLUSIONS: Our findings provide new insights into the biological significance of sex hormone receptors in HDP cells.

Sensitivity of human dental pulp cells to eighteen chemical agents used for endodontic treatments in dentistry.

To determine the adverse effects against human dental pulp tissue, the sensitivity of human dental pulp cells (D824 cells) to 18 chemical agents used for endodontic treatments in dentistry was examined. The cytotoxicity, as determined by a decrease in colony-forming ability of cells treated with the chemical agents, increased as the concentration increased. As a quantitative measure of the cytotoxic effect, LC(50), the concentration which induces a 50% lethality, was extrapolated from the concentration-response curves. The rank of the chemical agents according to their cytotoxic effect (LC(50)) was sodium arsenite > formaldehyde > hydrogen peroxide > zinc oxide > thymol ≈ iodoform ≈ eugenol > guaiacol > ethylenediaminetetraacetic acid ≈ iodine > procaine > lidocaine ≈ chloramphenicol ≈ m-cresol > calcium hydroxide ≈ sodium hypochlorite ≈ phenol ≈ p-phenolsulfonic acid. To compare the cytotoxicity and the levels of apoptosis and mRNA expression of five genes related to the function of dental pulp tissue, D824 cells treated with the LC(50) concentrations of chemical agents were assayed by the TUNEL method and quantitative reverse transcription polymerase chain reaction analysis, respectively. The inducibility of apoptotic cells and the level of mRNA expression of the genes varied with the chemical agents, indicating that both effects occurred independent of the rank of cytotoxic effect of the chemical agents. The results not only provide information concerning cytotoxicity of various chemical agents to human dental pulp cells, but also show an insight into the diversity of the pharmacodynamic action of the chemical agents.

Induction of mRNA expression of osteogenesis-related genes by guaiacol in human dental pulp cells.

To investigate the stimulating effect of endodontic medications on the mRNA expression of some osteogenesis-related genes associated with reparative dentinogenesis and hard-tissue formation, human dental pulp cells (D824 cells) were treated with calcium hydroxide (Ca (OH)(2)), formocresol, or guaiacol. The effect on growth was determined by growth curves of D824 cells treated for 1-3 days with 0.03-0.3 mM Ca (OH)(2), 0.0007%-0.0014% formocresol, or 0.24-2.43 mM guaiacol. The mitotic activity of individual cells and the mRNA expression of the osteogenesis-related genes for alkaline phosphatase (ALP), type I collagen (COL-1), and bone sialoprotein (BSP) in the cells treated for 24 h with the same concentrations of the medications as described above were determined by colony-forming efficiency and by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, respectively. Cellular growth and mitotic activity were scarcely affected by Ca (OH)(2), but were significantly reduced by formocresol or guaiacol. The mRNA expression of the osteogenesis-related genes was little affected by Ca (OH)(2) or formocresol, but was significantly enhanced by guaiacol. The results indicate that guaiacol may stimulate the mRNA expression of genes associated with reparative dentinogenesis and hard-tissue formation in human dental pulp cells, suggesting that the novel property of guaiacol provides new insights into the utilization of guaiacol in endodontic therapy.

Immortalization of normal human gingival keratinocytes and cytological and cytogenetic characterization of the cells.

Most in vitro studies of oral carcinogenesis in human cells are carried out with oral keratinocytes immortalized by human papillomavirus type 16 DNA. However, because various etiological factors for oral cancer are known, it is important to establish new human keratinocyte cell lines useful for studying the mechanism of oral carcinogenesis. Normal human gingival keratinocytes in secondary cultures grown in serum-free medium were either transfected with origin (-) SV40 DNA or sequentially transfected with origin (-) SV40 DNA and human c-fos. The transfected cells were continually passaged and analyzed for cytological and cytogenetic characterizations. Four immortal cell lines were grown for over 1100 days in culture and maintained a vigorous growth for over 250 population doublings. They expressed SV40 T antigen, cytokeratins 8 and 18, and E-cadherin, and overexpressed the c-Fos protein. The immortal cell lines had telomerase activity but lacked transformed phenotypes on soft agar or in nude mice. Each cell line had nonrandom chromosomal abnormalities and minisatellite alterations. One of the immortal cell lines, NDUSD-1, retained the capability to deposit calcium, which was also demonstrated in normal human gingival keratinocytes by alizarin red staining, indicating the possibility that NDUSD-1 cells may retain some natural characteristics of normal gingival keratinocytes. Because the oral ectoderm plays an important role in tooth development, these immortal cell lines may be useful in various experimental models for investigations of oral biology and oral carcinogenesis.

Changes in expression of imprinted genes following treatment of human cancer cell lines with non-mutagenic or mutagenic carcinogens.

It remains possible that chemicals that act by mutagenic mechanisms as well as chemicals that do not induce gene mutations may affect epigenetic gene expression. To test the possibility, we investigated the ability of both types of chemicals to alter the expression of five imprinted genes, PEG3, SNRPN, NDN, ZAC and H19, using two human colon cancer cell lines and a human breast cancer cell line. The expression of imprinted genes was changed by some non-mutagenic and mutagenic carcinogens independent of their mutagenic activity. The genes most commonly exhibiting the changes in expression were SNRPN and PEG3. Alterations of the expression of NDN and ZAC were also observed in some conditions. Methylation-specific PCR and chromatin immunoprecipitation assays suggest the possibility that changes in the expression of SNRPN may be associated with DNA hypomethylation and histone acetylation of the promoters and euchromatinization of the heterochromatic domains of the promoters. Changes in expression of the imprinted genes, PEG3 and NDN, were also observed in cells immortalized by treatment of normal human fibroblasts with 4-nitroquinoline 1-oxide or aflatoxin B1. We previously demonstrated that expression of the cancer-related gene, INK4a, in these immortal cells was lost via epigenetic mechanisms. The results prove that, in cancer cells, some mutagenic or non-mutagenic carcinogens can epigenetically influence the transcription levels of imprinted genes and also suggest the possibility that some chemical carcinogens may have epigenetic carcinogenic effects in human cells.

Clastogenic activity of seven endodontic medications used in dental practice in human dental pulp cells.

Numerous and varied chemical agents are used for endodontic treatments in dental practice. Endodontic medications are administered directly to the teeth in relatively high concentrations and chemical agents applied to enamel or dentin can penetrate the dental pulp tissue and circulate through the body in the bloodstream. In the present study, to assess safety regarding mutagenicity, we investigated the ability of seven endodontic medications to induce chromosome aberrations in human dental pulp cells. Chromosome aberrations were induced in cells treated with each of six endodontic medications, eugenol, guaiacol, modified phenol, phenol, thymol, and zinc oxide. The other endodontic medication, zinc chloride, failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by any of the endodontic medications tested. Our results indicate that the endodontic medications that exhibited a positive response are potentially mutagenic to human cells.

Ability of root canal antiseptics used in dental practice to induce chromosome aberrations in human dental pulp cells.

Root canal antiseptics are topically applied to root canals within the pulpless teeth to treat the root canal and periapical infections. Because the antiseptics that are applied to root canals can penetrate through dentin or leak out through an apical foramen into the periodontium and distribute by the systemic circulation, it is important to study the safety of these antiseptics. In the present study, we examined the ability to induce chromosome aberrations in human dental pulp cells of five root canal antiseptics, namely, carbol camphor (CC), camphorated p-monochlorophenol (CMCP), formocresol (FC), calcium hydroxide, and iodoform which are most commonly used in dental practice. Statistically significant increases in the levels of chromosome aberrations were induced by CC, FC, or iodoform in a concentration-dependent manner. Conversely, CMCP and calcium hydroxide failed to induce chromosome aberrations in the absence or presence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were enhanced by FC or iodoform. Our results indicate that the root canal antiseptics that exhibited a positive response are potentially genotoxic to human cells.

Assessment using human dental pulp cells of clastogenicity of antiseptics used in dental practice and agents for root canal enlargement and cleaning.

Numerous and varied chemical agents are used as topically applied drugs in dental practice. As they are administered directly to the oral cavity, it is important to study the safety of these agents. In the present study, to assess safety regarding mutagenicity, we investigated the abilities of six antiseptics to induce chromosome aberrations in human dental pulp cells. The antiseptics tested were benzalkonium chloride, benzethonium chloride, iodine glycerin, iodine tincture, oxydol, and povidone-iodine. In addition, we tested two agents used for root canal enlargement and cleaning, ethylenediaminetetraacetic acid and sodium hypochlorite. Chromosome aberrations were induced only in cells treated with the highest concentration of iodine tincture for 30 h. The other chemical agents failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The concentration of iodine tincture to which patients are exposed in dental practice is 1000-fold the concentration that induced chromosome aberrations in the present study. Our findings suggest that iodine tincture is mutagenic to human cells.

Effects of a catechol-O-methyltransferase inhibitor on catechol estrogen-induced cellular transformation, chromosome aberrations and apoptosis in Syrian hamster embryo cells.

To examine a possible mechanism of endogenous estrogen-induced carcinogenesis, we studied the effect of the catechol-O-methyltransferase (COMT) inhibitor Ro 41-0960 on cell transforming and clastogenic activities of 2 catechol estrogens 2- and 4-hydroxyestrone (2- or 4-OHE1) using Syrian hamster embryo (SHE) cells. COMT activity was assayed by determining the methylation of 2- or 4-OHE1 using gas chromatography. The production of 2-methoxyestrone in cultures treated with 2-OHE1 was approximately 2-fold that of 4-methoxyestrone in cultures treated with 4-OHE1. 4-OHE1 induced morphological transformation at a higher frequency than 2-OHE1 did and the frequencies of cell transformation and chromosome aberrations were not significantly changed in cells treated with 4-OHE1 in the presence of Ro 41-0960. In contrast, the frequencies of cell transformation and chromosome aberrations were markedly increased in cells treated with 2-OHE1 along with Ro 41-0960 when compared to cells treated with 2-OHE1 alone. In addition, both catechol estrogens induced P53 protein expression and apoptosis. The frequencies of apoptotic cells induced by the catechol estrogens were modified by the COMT inhibition in a manner similar to those observed with the chromosome aberrations assay and the cell transformation assay, indicating that each effect by the catechol estrogens at the three measured endpoints might be caused by a mechanism similar to the others. Our findings indicate that COMT activity has an influence on cell transforming activity and its related genetic effects of catechol estrogens in SHE cells, which implies that an individual activity of COMT may be one of the etiological factors in endogenous estrogen-induced carcinogenesis.

In vitro chromosome aberration tests using human dental pulp cells to detect the carcinogenic potential of chemical agents.

To examine if human dental pulp cells are useful for assessing the carcinogenic potential of chemical agents, we cultured human dental pulp cells from adults and studied the ability of chemical agents known to be carcinogenic to induce chromosome aberrations in these cells. We confirmed that human dental pulp cells in primary or secondary cultures had the capability of accumulating calcium in vitro as detected by Alizarin red staining and generating dentin-like tissue in immunocompromised mice. These phenotypes were maintained even in cells at seven passages. Next, we examined if chromosome aberrations were induced by exposure of human dental pulp cells (designated here as D824 cells) at seven to nine passages to chemical agents with carcinogenic activity. Statistically significant increases in the frequencies of chromosome aberrations were induced in D824 cells treated with a direct-acting carcinogen, mitomycin C, for 3 h. Chromosome aberrations were also induced at statistically significant levels in D824 cells treated with an indirect-acting carcinogen, cyclophosphamide, for 2 h in the presence of exogenous metabolic activation with rat liver postmitochondrial supernatant. Cyclophosphamide failed to induce chromosome aberrations in the absence of exogenous metabolic activation. Although the reliability of chromosome aberration tests using human dental pulp cells remains to be validated by studying the ability of various other chemical agents with or without carcinogenic activity to induce chromosome aberrations, this chromosome aberration test system may be useful for carcinogenic risk assessment in the target cells.

Reversible conversion of immortal human cells from telomerase-positive to telomerase-negative cells.

Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel(+)) to telomerase-negative (Tel(-)) cells. Tel(+) cells converted spontaneously to Tel(-) cells that reverted to Tel(+) cells following treatment with trichostatin A (TSA) and/or 5-aza-2'-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel(-) E6 Cl 6 cells. Tel(-) E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for >240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel(-) E6 Cl 6 cells and ALT cell lines were greater than those in Tel(+) cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.

Dmp1-deficient mice display severe defects in cartilage formation responsible for a chondrodysplasia-like phenotype.

Understanding the molecular mechanisms by which cartilage formation is regulated is essential toward understanding the physiology of both embryonic bone development and postnatal bone growth. Although much is known about growth factor signaling in cartilage formation, the regulatory role of noncollagenous matrix proteins in this process are still largely unknown. In the present studies, we present evidence for a critical role of DMP1 (dentin matrix protein 1) in postnatal chondrogenesis. The Dmp1 gene was originally identified from a rat incisor cDNA library and has been shown to play an important role in late stage dentinogenesis. Whereas no apparent abnormalities were observed in prenatal bone development, Dmp1-deficient (Dmp1(-/-)) mice unexpectedly develop a severe defect in cartilage formation during postnatal chondrogenesis. Vertebrae and long bones in Dmp1-deficient (Dmp1(-/-)) mice are shorter and wider with delayed and malformed secondary ossification centers and an irregular and highly expanded growth plate, results of both a highly expanded proliferation and a highly expanded hypertrophic zone creating a phenotype resembling dwarfism with chondrodysplasia. This phenotype appears to be due to increased cell proliferation in the proliferating zone and reduced apoptosis in the hypertrophic zone. In addition, blood vessel invasion is impaired in the epiphyses of Dmp1(-/-) mice. These findings show that DMP1 is essential for normal postnatal chondrogenesis and subsequent osteogenesis.

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow.

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.

Immortal, telomerase-negative cell lines derived from a Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities.

Five immortal cell lines derived from a Li-Fraumeni syndrome patient (MDAH 087) with a germline mutant p53 allele were characterized with respect to telomere length and genomic instability. The remaining wild-type p53 allele is lost in the cell lines. Telomerase activity was undetectable in all immortal cell lines. Five subclones of each cell line and five re-subclones of each of the subclones also showed undetectable telomerase activity. All five immortal cell lines exhibited variability in the mean length of terminal restriction fragments (TRFs). Subclones of each cell line, and re-subclones of the subclones also showed TRF variability, indicating that the variability is owing to clonal heterogeneity. Chromosome aberrations were observed at high frequencies in these cell lines including the subclones and re-subclones, and the principal types of aberrations were breaks, double minute chromosomes and dicentric chromosomes. In addition, minisatellite instability detected by DNA fingerprints was observed in the immortal cell lines. However, all of the cell lines were negative for microsatellite instability. As minisatellite sequences are considered recombinogenic in mammalian cells, these results suggest that recombination rates can be increased in these cell lines. Tumor-derived human cell lines, HT1080 cells and HeLa cells that also lack p53 function, exhibited little genomic instability involving chromosomal and minisatellite instabilities, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.