EXTERNAL AND INTERNAL EXPOSURE TO FUKUSHIMA RESIDENTS.

The Great East Japan Earthquake of 11 March 2011, caused the Fukushima Daiichi Nuclear Power Plant Accident, which resulted in the release of a large amount of radioactive materials into the environment, and there is a serious concern about the radiation effects on the health of residents living in the affected areas. The evaluation of exposure dose is fundamental for the estimation of health effects, and whenever possible, the exposure dose should be evaluated by actual measurements as opposed to estimations. Here, the outline of the exposure doses of residents estimated from surveys or obtained by measurements is described. Fukushima Health Management Survey reported the results for 460 408 residents during the first 4 months after the accident; 66.3% received doses <1 mSv, 94.9% received <2 mSv, 99.7% received <5 mSv and the maximum dose was 25 mSv. Thus, it was demonstrated that the results from personal dosemeter measurements were comparable to the estimations. The dose assessment of internal exposure of 184 205 residents conducted by Fukushima Prefecture by using whole body counter showed that 99.986% received <1 mSv, with the maximum dose being 3 mSv. Regarding exposure of the thyroid, there is not enough data for the Fukushima accident, but it is presumed that thyroid doses are much lower than those from Chernobyl. The outline of exposure doses of residents in result of the accident is still being clarified, questions and uncertainties in dose assessment remain and further efforts for more accurate dosimetry are required continuously.

Radiation Exposure and Thyroid Cancer Risk After the Fukushima Nuclear Power Plant Accident in Comparison with the Chernobyl Accident.

The actual implementation of the epidemiological study on human health risk from low dose and low-dose rate radiation exposure and the comprehensive long-term radiation health effects survey are important especially after radiological and nuclear accidents because of public fear and concern about the long-term health effects of low-dose radiation exposure have increased considerably. Since the Great East Japan earthquake and the Fukushima Daiichi Nuclear Power Plant accident in Japan, Fukushima Prefecture has started the Fukushima Health Management Survey Project for the purpose of long-term health care administration and medical early diagnosis/treatment for the prefectural residents. Especially on a basis of the lessons learned from the Chernobyl accident, both thyroid examination and mental health care are critically important irrespective of the level of radiation exposure. There are considerable differences between Chernobyl and Fukushima regarding radiation dose to the public, and it is very difficult to estimate retrospectively internal exposure dose from the short-lived radioactive iodines. Therefore, the necessity of thyroid ultrasound examination in Fukushima and the intermediate results of this survey targeting children will be reviewed and discussed in order to avoid any misunderstanding or misinterpretation of the high detection rate of childhood thyroid cancer.

Autologous adipose-derived regenerative cells are effective for chronic intractable radiation injuries.

Effective therapy for chronic radiation injuries, such as ulcers, is prone to infection. Stiffness is expected since the therapeutic radiation often involves wider and deeper tissues and often requires extensive debridement and reconstruction, which are not sometimes appropriate for elderly and compromised hosts. Autologous adipose-derived regenerative cells (ADRCs) are highly yielding, forming relatively elderly aged consecutive 10 cases, 63.6±14.9 y (52-89 y), with mean radiation dose of 75.0±35.4 Gy (50-120 Gy) were included with at least 10-month follow-up. Minimal debridement and ADRC injection in the wound bed and margin along with the injection of mixture of fat and ADRCs in the periphery were tested for efficacy and regenerated tissue quality by clinically as well as imaging by computed tomography and magnetic resonance imaging. Uncultured ADRCs of 1.6±1.3×10(7) cells were obtained. All cases healed uneventfully after 6.6±3.2 weeks (2-10 weeks) post-operatively. The done site morbidity was negligible and without major complications, such as paralysis or massive haematoma. The regenerated tissue quality was significantly superior to the pre-operative one and the mixture of fat and ADRCs connected to the intact tissue was very soft and pliable. Mean follow-up at 1.9±0.8 y (0.9-2.9 y) revealed no recurrence or new ulceration after treatment. Thus, the ADRCs treatment for decades-long radiation injuries is effective, safe and improves the quality of wounds.

Inhibition of ABL tyrosine kinase potentiates radiation-induced terminal growth arrest in anaplastic thyroid cancer cells.

Gleevec, a selective tyrosine kinase inhibitor, retarded the growth of anaplastic thyroid cancer cell lines in vitro and in vivo through selective inhibition of ABL tyrosine kinase activity. In the present study, we investigated the ability of Gleevec to modulate the in vitro and in vivo radiation response of anaplastic thyroid cancer cells. Cell growth assays, colony formation assays and xenograft models were used to quantify the radiosensitizing effect of Gleevec in cells of the anaplastic thyroid cancer cell lines ARO and FRO. FACS, Western blotting and histochemical techniques were employed to study the mechanisms of radiation response after exposure to Gleevec. Gleevec (7.0 microM) increased the anti-proliferative effect of radiation on the growth ARO and FRO cells in vitro. Clonogenic analysis demonstrated that Gleevec reduced cell survival after irradiation. Gleevec combined with radiation produced an increase in tumor growth inhibition compared to treatment with either modality alone in mice bearing anaplastic thyroid cancer xenografts. The drug suppressed radiation-induced ABL activation and promoted CDKN1A (p21(cip1)) accumulation in irradiated anaplastic thyroid cancer cells. Gleevec had an additional effect on radiation-induced apoptosis in cells of both cell lines and potentiated the induction of terminal growth arrest accompanied by the expression of senescence-associated beta-galactosidase. The antitumor effect of Gleevec is potentiated in adjunctive therapy with radiation not only due to inhibition of proliferative cell growth with transient cell cycle arrest and apoptosis, but also due to the terminal growth arrest associated with senescence, suggesting that tumor cell senescence is a mechanism for tumor targeting therapy in combination with ionizing radiation.

Eradication of hepatocellular carcinoma xenografts by radiolabelled, lipiodol-inducible gene therapy.

The promoter region of the early-growth response-1(Egr-1) gene has been shown to be activated by external radiation, thus making a selective tumoricidal effect possible. A previous experiment showed that the Egr-1 promoter can be activated by internal radiation using radioisotopes as well as external radiation. Internal radiation using I-131 lipiodol (I-131-Lip) has been established as one of the most useful therapeutic strategies against hepatoma. We herein linked the Egr-1 promoter to the herpes simplex virus-thymidine kinase (HSV-TK) gene, and investigated its efficacy in hepatoma gene therapy in combination with I-131-Lip. A luciferase assay showed the Egr-1-promoter activity to be markedly increased in hepatoma tissue specimens in an I-131-dose-dependent manner, whereas a less than two-fold increase in this activity was observed in other organs. In addition, the radioactivity derived from I-131 was selectively accumulated in the tumor tissue specimens. To examine the efficacy of EgrTK/ganciclovir (GCV) gene therapy in vivo, subcutaneous hepatoma xenografts in nude mice were transfected using a hemagglutinating virus of Japan (HVJ)-liposome vector. Complete tumor regression was observed in all the EgrTK-transfected tumors following combination treatment with I-131-Lip and GCV 42 days after treatment without any side effects (n=8). In contrast, the tumors continued to grow in all control mice (n=10). Furthermore, the serum alpha-fetoprotein levels decreased in the combination therapy group, while they increased in the controls. In conclusion, these data indicate that Egr-1 promoter-based gene therapy combined with internal radiation has a selective effect on hepatoma tumors while also showing an improved in vivo efficacy. This combination therapy might, therefore, be an effective human hepatoma gene therapy, even in advanced multiple cases.

Expression of Tie-1 and 2 receptors, and angiopoietin-1, 2 and 4 in gastric carcinoma; immunohistochemical analyses and correlation with clinicopathological factors.

AIMS: There is strong evidence that tyrosine kinases are involved in the regulation of tumour progression, cellular growth and differentiation. Recently, many kinds of tyrosine kinase receptors have been reported, and among them Tie-1 and 2 constitute a major class. Angiopoietin (Ang)-1 is known as a ligand of the Tie-2 tyrosine kinase receptor. The aim of this study was to determine the expression profile of Tie-1 and 2 and Ang-1, 2 and 4 in gastric adenocarcinoma. METHODS AND RESULTS: Eighty-nine cases of surgically resected human gastric adenocarcinoma were studied by immunohistochemistry. Of these, 60 (67.4%), 61 (68.5%), 69 (77.5%), 75 (84.3%), and 47 cases (52.8%) showed positive staining in the cytoplasm of carcinoma cells for the Tie-1 and 2 and Ang-1, 2 and 4 proteins, respectively. The expression of Ties and Angs was significantly correlated with several type of histological differentiation and several clinicopathological factors. CONCLUSIONS: Ties and Angs were highly expressed in human gastric adenocarcinoma cells. These findings suggest that the Tie-Ang receptor-ligand complex is one of the factors involved in the cellular differentiation and progression of human gastric adenocarcinoma.

Radiation-inducible hSNK gene is transcriptionally regulated by p53 binding homology element in human thyroid cells.

We identified a species relevant to polo-like kinase family, a human homologue of mouse serum-inducible kinase, hSNK gene, whose mRNA expression was rapidly increased in cultured human thyroid cells after X-ray irradiation. The cDNA cloning and genomic analysis of the hSNK gene showed the presence of 14 exons spanning over 6 kb of genomic DNA that encodes a 2.9-kb mRNA product. Promoter analysis demonstrated possible existence of a radiation-responsive element in the p53 binding homology element (p53RE) localized to near upstream of basal promoter of the hSNK gene. Nuclear protein extracts from HeLa and various human thyroid carcinoma cell lines bound selectively to p53RE. Anti-p53 or anti-p73 antibodies, however, failed to recognize the p53RE-protein complex formed in the presence of such nuclear extracts. These results suggest that radiation-responsive transcription factor(s) directly participates in the regulation of hSNK gene expression via the binding to p53RE in promoter region.

Transient activation of c-Jun NH2-terminal kinase by growth factors influences survival but not apoptosis of human thyrocytes.

Activation of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, is involved in apoptosis or cell proliferation. We have previously demonstrated that ionizing radiation or thyroid-stimulating hormone activated JNK without linking to thyroid cell apoptosis. To clarify the involvement of JNK activation in thyroid cell survival, we investigated the effects of various growth factors on induction of JNK activation in cultured human thyroid cells. JNK activation was observed at 30 minutes after fetal bovine serum (FBS) stimulation and returned to basal level at 240 minutes. Epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF) also induced JNK activation, but did not trigger apoptotic cell death. Furthermore, we observed high basal activation of JNK in four of five human thyroid cancer cell lines. Overexpression of c-Met, an HGF receptor, was observed in two of the four cell lines with high basal JNK activity. Our results suggest that JNK activation does not induce apoptosis but is associated with survival or transformation of human thyroid cells.

Expression of the ets-1 proto-oncogene in human colorectal carcinoma.

The proto-oncogene, ets-1, is a transcription factor known to control the expression of a number of genes involved in extracellular matrix remodeling and has been postulated to play a role in cell migration and tumor invasion. To elucidate the involvement of ets-1 in human colorectal carcinomas, we examined 41 cases of colorectal adenoma and 122 cases of colorectal carcinoma by immunohistochemistry and compared the degree of Ets-1 expression with the depth of carcinoma invasion. In adenomas, 12 of 41 cases (29.3%) showed immuno-positivity for Ets-1. 12 of 27 cases (44.4%) of adenoma with high grade dysplasia showed immunopositivity for Ets-1. However, there was no positive case in low or moderate dysplasia of adenoma. In contrast, 103 of 122 cases (84.4%) of colorectal adenocarcinoma showed immunoreactivity for Ets-1 in the carcinoma cells themselves. We investigated the relationship between pathological features in colorectal carcinoma and Ets-1 immunoreactivity of the tumor cells. Among the 122 cases of invasive carcinomas, Ets-1 immunoreactivity was significantly correlated with the depth grading of tumor invasion (P < .0001), the presence of lymph node metastasis (P < .05), lymphatic invasion (P < .01) and venous invasion (P < .05). However, Ets-1 expression did not correlate with histological differentiation. In situ hybridization also confirmed the presence of ets-1 mRNA in colorectal carcinomas. Expression of ets-1 mRNA was also detected in two of three human colorectal carcinoma tissues and in four of six different kinds of carcinoma cell lines by the reverse transcription polymerase chain reaction method. These findings suggest that the expression of Ets-1 is one of the important factors related to carcinogenesis and/or tumor invasion of colorectal carcinoma.

PKC delta mediates ionizing radiation-induced activation of c-Jun NH(2)-terminal kinase through MKK7 in human thyroid cells.

The thyroid gland is one of the most sensitive organs in ionizing radiation (IR)-induced carcinogenesis. To determine, therefore, the specific cascade of IR-induced signal transduction in human thyroid cells, we investigated the functional role of protein kinase C (PKC), especially its interlocking activation of c-Jun NH(2)-terminal kinase (JNK) pathway. In the present study, using adenovirus expression vectors for diverse dominant-negative (DN) types of PKC isoforms (alpha, beta2, delta, epsilon and zeta) expressed in primary cultured human thyroid cells, only DN/PKC delta suppressed IR-induced JNK activation. In addition, Rottlerin, a PKC delta specific inhibitor, inhibited IR-induced JNK activation. IR-induced activation of transcription factor AP-1, downstream target of JNK, was also attenuated by DN/PKC delta. To examine the involvement of upstream kinases of JNK, we performed immune-complex kinase assays of mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. IR activated MKK7 but not MKK4, and this activation was inhibited by Rottlerin. Furthermore, IR-induced JNK activation was suppressed by overexpression of kinase-deficient MKK7. Our results indicate that IR selectively activates the cascade of PKC delta-MKK7-JNK-AP-1 in human thyroid cells, suggesting a not apoptotic but radio-resistant role of PKC delta in human thyroid cells following IR.

The sympathetic nervous system promotes carbon tetrachloride-induced liver cirrhosis in rats by suppressing apoptosis and enhancing the growth kinetics of regenerating hepatocytes.

Norepinephrine is considered to possess potent anti-apoptotic action in regenerating hepatocytes. To clarify the role of the sympathetic nervous system in apoptosis that occurs in chronic liver damage and following the promotion of liver cirrhosis, we studied a carbon tetrachloride (CCl4)-induced liver injury model, using spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and chemically sympathectomized WKY. At 24 h after CCl4 administration. acute damage, characterized by vacuolated hepatocytes in the centrilobular zone, was greater in SHR than in WKY. This vacuolated change in WKY hepatocytes was significantly reduced by chemical sympathectomy with 6-hydroxydopamine (6-OHDA). After 48 h, the acute damage was dramatically improved in each animal, without significant differences between the three groups. In chronic damage after weekly repetition of CCl4 treatment for 4 weeks, fibrosis was evident in SHR, while in the other groups there was only scant fibrosis in the centrilobular zone. After 8 weeks' repetition of CCl4, liver cirrhosis was seen only in SHR. The incidence of apoptotic cells in areas of both acute and chronic damage in WKY, detected by terminal deoxynucleotidyl transferase-dUTP nick end labeling, was significantly increased in comparison with that in SHR, and was further increased by 6-OHDA pretreatment. In contrast, there was significantly greater enhancement of the growth of hepatocytes in SHR than in WKY in both acute and chronic damage. Moreover. hepatocyte growth kinetics in WKY was significantly inhibited after sympathectomy in acute injury, as evidenced by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In vitro, the amount of hepatocellular apoptosis induced by transforming growth factor-beta1 was significantly decreased by incubation with norepinephrine. These findings suggest that the anti-apoptotic effect of the sympathetic nervous system increases cell growth kinetics and promotes liver cirrhosis in this animal model.

Keloid fibroblasts resist ceramide-induced apoptosis by overexpression of insulin-like growth factor I receptor.

Keloids are benign dermal tumors, characterized by overgrowth of lesions, invasiveness beyond the original boundary of the insult, and recurrence of lesions. The exact etiology is unknown, however. Our hypothesis is that keloids are acquired as a result of an abnormal or prolonged wound healing process, with persistent proliferation and extracellular matrix production of fibroblasts that should otherwise discontinue in normal wound healing. In this study, we examined the response of keloid fibroblasts to proapoptotic signaling. Cell-permeable ceramide, N-acetyl-D-sphingosine, induced apoptosis of dermal fibroblasts in a dose- and time-dependent manner, which was detected by phase contrast microscopy, fluorescent microscopy, the TUNEL method, flow cytometric analysis, and WST-1 assay. In contrast, keloid fibroblasts resisted apoptosis induced by N-acetyl-D-sphingosine (percent survival with 40 mM ceramide treatment for 12 h, normal versus keloid: 9.6% +/- 6.6% vs 66.8% +/- 5.5%). Western blotting analysis showed insulin-like growth factor I receptor overexpression in keloid fibroblasts, but not in normal fibroblasts. Exogenously added insulin-like growth factor I enhanced the resistance of keloid fibroblasts to ceramide-induced apoptosis. Wort- mannin, a phosphatidylinositol 3 kinase inhibitor, suppressed the antiapoptotic action of insulin-like growth factor I in keloid fibroblasts. Our results suggest that keloid fibroblasts overexpressing insulin-like growth factor I receptor are resistant to apoptosis, thus allowing persistent proliferation and production of excessive extracellular matrix. J Invest Dermatol 115:1065-1071 2000

Safe and efficient gene transfer into porcine hepatocytes using Sendai virus-cationic liposomes for bioartificial liver support.

Establishment of a bioartificial liver support system using genetically modified hepatocytes is a potential approach to improve the treatment of severe liver failure. We describe the development of an efficient ex vivo method of gene transfer into a large number of porcine hepatocytes using hemagglutinating virus of Japan (HVJ)-liposome. The transfection efficiency of HVJ-liposome into isolated porcine hepatocytes attached to microcarrier beads was evaluated by beta-galactosidase (beta-gal) staining, fluorescence activated cell sorting analysis for beta-gal and luciferase assay, respectively. To examine the function and cellular damage of transduced hepatocytes, we used enzyme-linked immunosorbent assay for porcine albumin synthesis, lidocaine clearance test (P-450 activity), aspartate aminotransferase, and lactic dehydrogenase release assays. The optimal conditions for gene transfer into the beads-attached hepatocytes using HVJ-liposome included 4 microg of deoxyribonucleic acid with 200 microg of lipid/2 x 105 cells and exposure duration of 90 min. Under these conditions, beta-gal and luciferase genes were transduced to 2.5 x 108 isolated porcine hepatocytes following attachment to the beads. Positive beta-gal staining was observed in more than 30% of the beads-attached hepatocytes. The gene transfer activity of HVJ-liposome method determined by luciferase activities was about 100-fold of that of the lipofection method. Transfected porcine hepatocytes remained functional without any significant cell damage. Our results demonstrated that HVJ-liposome mediated gene transfer into microcarrier-attached porcine hepatocytes is an efficient and nontoxic method suitable for a bioartificial liver support sytem.

Eradication of breast cancer xenografts by hyperthermic suicide gene therapy under the control of the heat shock protein promoter.

To investigate the usefulness of heat shock protein (HSP) promoter for breast cancer gene therapy, hyperthermia and HSV thymidine kinase (tk) suicide gene combination therapy was examined with mouse mammary cancer cell line FM3A. HSP promoter activity was markedly increased after heat shock (41-45 degrees C), with maximum activation (about 400-fold) at 3 hr. An in vitro cytotoxic assay showed that HSP-tk-transduced FM3A cells became more sensitive (more than 50,000 times) to ganciclovir (GCV) with heat shock, but untreated cells showed no increased cytotoxic sensitivity to GCV compared with control FM3A cells. In addition to promoter-oriented selective cell killing, a "chemosensitization effect" as a bystander effect was demonstrated by hyperthermia and suicide gene combination therapy, using a non-heat-inducible promoter. Immunohistochemical analysis revealed that this synergistic killing effect was dependent on apoptotic cell death with upregulation of both Fas and FasL (Fas ligand) expression. We also examined the efficacy of HSP-tk gene therapy in vivo by implanting breast cancer in subcutaneous and intraperitoneal models of BALB/c nude mice targeted by the HVJ-anionic liposome method. Significant tumor regression was observed in HSP-tk-transduced tumors followed by hyperthermia therapy, but no such inhibition was noted in either the mock vector transfection or hyperthermia group compared with control tumor-bearing mice. Our results demonstrate that this combination system is synergistically effective in mediating Fas-dependent apoptosis for a specific gene therapy targeting HSP-expressing mammary carcinomas, even in advanced and heat-resistant breast cancer.

Parathyroid hormone-related peptide is a potent tumor angiogenic factor.

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivo diffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 microM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1-34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1-34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.

Sage transcript profiles in cultured human fetal fibroblasts, WI-38.

Serial analysis of gene expression (SAGE) allows us to analyze the profile of genes expressed in human lung fibroblast cell line WI-38. Manual sequencing of approximately 10,000 transcripts derived from WI-38 cells revealed 1025 genes expression among of which 431 tags matched GenBank entries, whereas 594 corresponded to novel previously undescribed transcripts. 7SL RNA (7L7) 5'-truncated pseudogene and the gene with no match (tag: TCCCTAGCT) were found the most abundant among all the analyzed genes (10.2 and 4.6% respectively). The expression pattern of the genes coding for cytoskeletal proteins, extracellular matrix components (ECM) and cytokines has been obtained. The results here demonstrate the unique gene expression pattern in human WI-38 fibroblast cell line and support a possibility of useful application of SAGE method for differential expression analysis.

Ceramide-induced apoptosis of human thyroid cancer cells resistant to apoptosis by irradiation.

Ionizing radiation (IR) induces apoptosis through, in part, cell membrane breakdown signals. Ceramide and diacylglycerol (DAG) are released after IR exposure, which act as second messengers to induce proapoptotic and antiapoptotic signals, respectively. We have previously shown, however, that thyroid cells are relatively resistant to IR-induced apoptosis. To investigate the mechanism of thyroid cell resistance to IR-related apoptosis, we determined the effects of ceramide and its release following exposure of human thyroid cancer cell lines to IR. Exogenous C2-ceramide (10-100 microM) activated the apoptosis process in all cell lines used. Exogenous C2-ceramide also activated a stress kinase, c-Jun N-terminal kinase UNK). The apoptotic action of ceramide was attenuated by serum or simultaneous activation of protein kinases C and A by phorbol esters and forskolin. Furthermore, 2-5 Gy IR had a differential effect on ceramide and DAG release in human thyroid cells; a weak and transient release of ceramide but a strong and sustained release of DAG. Our results indicated that the radioresistance properties of thyroid cancer cells probably reflect the dominance of anti-apoptotic signals, evoked by growth factor(s) and DAG, which override the apoptotic effect of ceramide released by human thyroid cells on exposure to IR, in spite of activation of proapoptotic pathway downstream of ceramide.

Insulin-like growth factor-I (IGF-I)/IGF-I receptor axis and increased invasion activity of fibroblasts in keloid.

Activation of signals for insulin-like growth factor-I receptor (IGF-IR) is thought to be closely linked to abnormal cell proliferation and differentiation in various diseases. The keloid in which fibroblasts invade beyond the margins of the original wound, is a dermal fibroproliferative tissue of unknown etiology. Clinically, keloids are most commonly observed in subjects at ages between 10 and 30 years. Interestingly, plasma levels of growth hormone and IGF-I are also high during the same period, suggesting that IGF-I might be involved in the patho-physiology of keloid fibroblasts. We therefore first examined the expression level of IGF-IR in normal and keloid tissues. Immunohistochemical analysis confirmed increased expression of IGF-IR in keloid fibroblasts, but not in normal fibroblasts. On the other hand, the staining intensity of IGF-IR in the epidermis of normal tissues was almost equal to that in keloids. Next, to study the functional properties of the IGF-I/IGF-IR axis in both normal and keloid fibroblasts, we investigated invasion activities. The invasive activity of IGF-IR overexpressing keloid fibroblasts was greatly increased in the presence of IGF-I, and inhibited by a neutralizing antibody to IGF-I. In contrast, its activity of IGF-IR weak-expressing normal fibroblasts was not changed. Our results indicate the involvement of the activated IGF-I/IGF-IR axis in the pathogenesis of the invasive activity of fibroblasts.

Transforming growth factor-beta stimulates articular chondrocyte cell growth through p44/42 MAP kinase (ERK) activation.

Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-beta1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-beta1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-beta1, we performed in vitro kinase assays using specific substrates. TGF-beta1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240 min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-beta1-induced cell growth by 40%. These results indicate that TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-beta1.