The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A 12S in response to dexamethasone. The level of topoisomerase II␣ begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncogene 10, 651-662). Topoisomerase II␣ prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP-and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase II␣ was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 ؋ g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase II␣ could be detected by incubating it in the S70 or S100 extract, which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42-h extract was 4-to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component(s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase II␣, is activated or induced during the latent phase of E1A-induced apoptosis.

The human epidermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the hormone-inducible promoter, elicits apoptosis after induction of E1A 12 S in response to dexamethasone. E1A expression caused accumulation of wild type p53 more than 10-fold within 24 h after dexamethasone treatment. The cell lines that express E1A mutants containing a deletion either in the amino terminus or the conserved region 1 were unable to accumulate p53. p53 accumulated was degraded efficiently in vitro in the S10 -0 extract (S10 -0) prepared from MA1 cells in an ATP and ubiquitin-dependent manner, but not in S10 -24 prepared after treatment with dexamethasone for 24 h. The p53 polyubiquitination activity in S100 -0 was calcium-dependent and reduced greatly in S100 -24. Ubiquitin affinity chromatography revealed that p53 ubiquitination activity in eluates thought to contain ubiquitin-conjugating enzymes decreased greatly in S100 -24 as compared with S100 -0. The accumulation of p53 was accompanied by the increase in the level of Mdm2, which has been shown to degrade p53 through binding to it. The high p53 level, however, was maintained until the late stage of the apoptotic process. These results indicate that the stabilization of p53 by E1A occurs through modification of a ubiquitin-specific enzyme(s) in the ubiquitin-proteasome pathway.

The rat 3Y1 derivative cell lines, EId10 and EId23, established by introducing the adenovirus E1A12S, Id-1H, and Id-2H cDNAs linked to the hormone-inducible promoter, express these proteins upon treatment with dexamethasone and elicit apoptosis, although these cell lines express mutated p53. The E1A mutants containing a deletion in either the N terminus or the conserved region 1 were unable to induce apoptosis in cooperation with Ids. Western blot analysis of the immunoprecipitates prepared from the dexamethasone-treated EId10 cell extract showed that Id-2H preferentially binds to E1A and E2A (E12͞E47) helix-loop-helix transcription factors in vivo, but scarcely to the retinoblastoma protein. After induction of E1A and Ids, EId10 and EId23 cells began to accumulate in S phase and undergo apoptosis before entering G 2 phase, suggesting that abnormal synthesis of DNA induced by coexpression of E1A, Id-1H, and Id-2H results in the induction of apoptosis. Apoptosis also is induced in mouse A40 (p53؊͞؊) cells by E1A alone or E1A plus Ids after transient transfection of the expression vectors. The induction of apoptosis is stimulated by coexpression with wild-type p53; however, apoptosis induced by E1A alone was suppressed completely by coexpression with mutated p53, whereas apoptosis induced by E1A plus Ids was stimulated by the mutated p53 as done by wild-type p53. These results suggest that the suppressive function of mutated p53 is overcome by Ids.The adenovirus E1A gene products (E1A) have functions to induce cell proliferation and death and to inhibit cell differentiation in a variety of cell types. The E1A gene of human adenovirus types 2 and 5 generates two major species of mRNA with the sizes of 13S and 12S that encode proteins of 289 aa (E1A 13S ) and 243 aa (E1A 12S ), respectively. E1A 13S contains conserved regions CR1, CR2, and CR3, but E1A 12S lacks CR3 (1). The N terminus of E1A 5Ј to CR1 interacts with basic helix-loop-helix (bHLH) transcription factors and inhibits terminal differentiation of muscle cells (2). As reported here, Id (inhibitor of differentiation or inhibitor of DNA binding) proteins (3), a family of HLH proteins lacking the basic domain, also binds to the N terminus of E1A. The E1A domain required for induction of p53-dependent apoptosis recently has been mapped in the N terminus and the CR1 regions (4, 5), which overlap with the p300 binding domain (1).bHLH proteins, such as MyoD and E2A (E12͞E47), bind to DNA efficiently when these proteins heterodimerize (3). Id proteins also heterodimerize with bHLH proteins through the HLH domain, suppressing their DNA binding activity (3). Expression levels of Id proteins decrease in a variety of undifferentiated cells, upon induction of differentiation (3). The cDNA clones of four members of the mouse and human Id families have been isolated. Id-1H and Id-2H isolated by us from a human cDNA library (6) correspond to mouse Id-1 and Id-2, respectively. Id-1H and Id-2H are induced in quiescent human diploid fibroblasts by serum factors biph...

Microtia is a congenital aplasia of the auricular cartilage. Conventionally, autologous costal cartilage grafts are collected and shaped for transplantation. However, in this method, excessive invasion occurs due to limitations in the costal cartilage collection. Due to deformation over time after transplantation of the shaped graft, problems with long-term morphological maintenance exist. Additionally, the lack of elasticity with costal cartilage grafts is worth mentioning, as costal cartilage is a type of hyaline cartilage. Medical plastic materials have been transplanted as alternatives to costal cartilage, but transplant rejection and deformation over time are inevitable. It is imperative to create tissues for transplantation using cells of biological origin. Hence, cartilage tissues were developed using a biodegradable scaffold material. However, such materials suffer from transplant rejection and biodegradation, causing the transplanted cartilage tissue to deform due to a lack of elasticity. To address this problem, we established a method for creating elastic cartilage tissue for transplantation with autologous cells without using scaffold materials. Chondrocyte progenitor cells were collected from perichondrial tissue of the ear cartilage. By using a multilayer culture and a three-dimensional rotating suspension culture vessel system, we succeeded in creating scaffold-free elastic cartilage from cartilage progenitor cells.

Peripheral blood neutrophils were isolated from the venous blood of healthy volunteers and aged in culture to provide a population of apoptotic neutrophils. Increased expression of PKCD and 6 isoeqmes was detected by Western blotting in nartrophils undergoing spontaneous apoptosis. Increased Bssociation of / 3 and 6 isoeqmes with the cell membrane fiaction, which is an indicator of PKC activation, was also

In this report we demonstrate the effect of a novel electron emission-based cell culture device on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells. Our device has an electron emission element that allows, for the first time, stable emission of electrons into an atmosphere. Atmospheric electrons react with gas molecules to generate radicals and negative ions, which induce a variety of biochemical reactions in the attached cell culture system. In this study, we demonstrated the effect of this new electron emission-based cell culture device on cell proliferation and differentiation using pre-osteoblastic MC3T3-E1 cells. Electron emission stimulation (EES) was applied directly to culture medium containing plated cells, after which the number of living cells, the mRNA levels of osteogenesis-related genes, and the alkaline phosphatase (ALP) activity were evaluated. The growth rate of EES-exposed cells increased by approximately 20% in comparison with unexposed control cells. We also found the mRNA levels of osteogenic specific genes such as collagen type I α-1, core-binding factor α-1, and osteocalcin to be up-regulated following EES. ALP activity, a marker for osteogenic activity, was significantly enhanced in EES-treated cells. Furthermore, reactive oxygen species generated by EES were measured to determine their effect on MC3T3-E1 cells. These results suggest that our new electron emission-based cell culture device, while providing a relatively weak stimulus in comparison with atmospheric plasma systems, promotes cell proliferation and differentiation. This system is expected to find application in regenerative medicine, specifically in relation to bone regeneration.