Sustained activation of the FGF1-MEK-ERK pathway inhibits proliferation, invasion and migration and enhances radiosensitivity in mouse angiosarcoma cells.

Angiosarcoma is a rare refractory soft-tissue tumor with a poor prognosis and is treated by radiotherapy. The fibroblast growth factor 1 (FGF1) mutant, with enhanced thermostability due to several substituted amino acids, inhibits angiosarcoma cell metastasis, yet the mechanism of action is unclear. This study aims to clarify the FGF1 mutant mechanism of action using ISOS-1 mouse angiosarcoma cells. The wild-type FGF1 or FGF1 mutant was added to ISOS-1 cells and cultured, evaluating cell numbers over time. The invasive and migratory capacity of ISOS-1 cells was assessed by transwell analysis. ISOS-1 cell radiosensitivity was assessed by colony formation assay after X-ray irradiation. To examine whether mitogen-activated protein kinase (MEK) inhibitor counteracts the FGF1 mutant effects, a combination of MEK inhibitor and FGF1 mutant was added to ISOS-1 cells and cultured. The FGF1 mutant was observed to inhibit ISOS-1 cell proliferation, invasion and migration by sustained FGF1 signaling activation. A MEK inhibitor suppressed the FGF1 mutant-induced inhibition of proliferation, invasion and migration of ISOS-1 cells. Furthermore, the FGF1 mutant enhanced radiosensitivity of ISOS-1 cells, but MEK inhibition suppressed the increased radiosensitivity. In addition, we found that the FGF1 mutant strongly inhibits actin polymerization, suggesting that actin cytoskeletal dynamics are closely related to ISOS-1 cell radiosensitivity. Overall, this study demonstrated that in ISOS-1 cells, the FGF1 mutant inhibits proliferation, invasion and migration while enhancing radiosensitivity through sustained activation of the MEK-mediated signaling pathway.

High-Sulfated Hyaluronic Acid Ameliorates Radiation-Induced Intestinal Damage Without Blood Anticoagulation.

Purpose: Many growth factors, such as fibroblast growth factors (FGFs), are useful for the treatment or prevention of radiation damage after radiation therapy. Although heparin can be supplemented to increase the therapeutic effects of FGFs, it possesses strong anticoagulant effects, which limit its potential for clinical use. Therefore, chemically sulfated hyaluronic acid (HA) was developed as a safe alternative to heparin. This study examined the involvement of sulfated HA in radioprotective and anticoagulant effects. Methods and Materials: FGF1 was administered intraperitoneally to BALB/c mice with sulfated HA 24 hours before or after total body irradiation with γ-rays. Several radioprotective effects were examined in the jejunum. The blood coagulation time in the presence of sulfated HA was measured using murine whole blood. Results: FGF1 with high-sulfated HA (HA-HS) exhibited almost the same level of in vitro mitogenic activity as heparin, whereas FGF1 with HA or low-sulfated HA exhibited almost no mitogenic activity. Furthermore, HA-HS had high binding capability with FGF1. FGF1 with HA-HS significantly promoted crypt survival to the same level as heparin after total body irradiation and reduced radiation-induced apoptosis in crypt cells. Moreover, pretreatment of HA-HS without FGF1 also increased crypt survival and reduced apoptosis. Crypt survival with FGF1 in the presence of HA depended on the extent of sulfation of HA. Moreover, the blood anticoagulant effects of sulfated HA were weaker than those of heparin. As sulfated HA did not promote the reactivity of antithrombin III to thrombin, it did not increase anticoagulative effects to the same extent as heparin. Conclusions: This study suggested that HA-HS promotes the radioprotective effects of FGF1 without anticoagulant effects. HA-HS has great potential for practical use to promote tissue regeneration after radiation damage.

Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions.

Human induced pluripotent stem (hiPS) cells are attracting attention as a tool for regenerative medicine. However, several problems need to be overcome for their widespread and safe use, for example, the high cost of maintaining hiPS cells and the possibility of xenogeneic cell contamination in hiPS cell cultures. One of the main contributors to the high cost of maintaining hiPS cells is basic fibroblast growth factor (bFGF), which is essential for such cultures. Xenogeneic contamination can occur because of the use of mouse-derived feeder cells to culture hiPS cells. To overcome the problems of cell culture cost and xenogeneic contamination, we have developed a novel culture method in which the undifferentiated state and pluripotency of hiPS cells can be maintained under feeder-free and bFGF-free conditions. Our new approach involves the addition to the culture medium of highly sulfated hyaluronic acid (HA-HS), in which the hydroxyl groups of d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) are chemically sulfated. HA-HS promotes bFGF signaling and maintains the undifferentiated state and pluripotency of hiPS cells under feeder-free and bFGF-free conditions. By contrast, non-sulfated hyaluronic acid and low sulfated hyaluronic acid do not maintain the undifferentiated state and pluripotency of hiPS cells. These results indicate that the maintenance of hiPS cells under feeder-free and bFGF-free conditions is an HA-HS specific effect. This study is the first to demonstrate the effects of sulfated hyaluronic acid on mammalian pluripotent stem cells, and provides a novel method for maintaining hiPS cells using HA-HS.

The FGF1/CPP-C chimera protein protects against intestinal adverse effects of C-ion radiotherapy without exacerbating pancreatic carcinoma.

BACKGROUND AND PURPOSE: Carbon ion (C-ion) beams are concentrated to irradiate pancreatic carcinoma in the upper abdomen; however, this radiotherapy potentially causes adverse reactions in the gastrointestinal tract. FGF1 is a candidate radioprotector for radiation-induced intestinal damage, but may promote the malignancy of pancreatic cancer. An FGF1/CPP-C chimeric protein was created to enhance the intracellular signaling mode of FGF1 instead of FGFR signaling. The present study investigated the effects of FGF1/CPP-C on the intestinal adverse reactions of C-ion radiotherapy as well as its influence on the malignancy of pancreatic cancer. MATERIALS AND METHODS: FGF1/CPP-C was administered intraperitoneally to BALB/c mice without heparin 12 h before total body irradiation (TBI) with low-LET C-ion (17 keV/µm) at 6-8 Gy. Several radioprotective effects were examined in the jejunum. The invasion and migration of the human pancreatic carcinoma cell lines MIAPaCa-2 and PANC-1 were assessed using Boyden chambers after cultures with FGF1/CPP-C. RESULTS: The FGF1/CPP-C treatment promoted crypt survival after C-ion irradiation at 7-8 Gy significantly more than the FGF1 treatment. FGF1/CPP-C also inhibited C-ion radiotherapy-induced apoptosis and reduced γH2AX foci in crypt cells more than FGF1. However, FGF1/CPP-C inhibited the downstream signaling pathways of FGFRs and suppressed the activation of cell-cycle regulatory molecules in the intestine until 4 h after TBI. Furthermore, IEC6 cells were arrested in G2M after cultures with FGF1/CPP-C or FGF1, suggesting that DNA repair after irradiation is promoted by FGF1/CPP-C-induced G2M arrest. In contrast, FGF1/CPP-C appeared to be internalized into MIAPaCa-2 and PANC-1 cells more efficiently than FGF1. Therefore, FGF1/CPP-C reduced the in vitro proliferation, invasion, and migration of MIAPaCa-2 and PANC-1 cells significantly more than FGF1 through the cellular internalization of FGF1. CONCLUSION: These results suggest that the intracellular signaling mode of FGF1/CPP-C attenuates the intestinal adverse effects of C-ion radiotherapy without enhancing the malignancy of pancreatic carcinoma.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Fra­1 enhances the radioresistance of colon cancer cells to X­ray or C­ion radiation.

Fos­related antigen 1 (Fra­1) has roles in a variety of cell functions, including cell proliferation, differentiation, transformation, and invasiveness, and it is upregulated in various cancers. We investigated the role of Fra­1 in cellular radioresistance using cells of two human colorectal cancer cell lines, SW620 and SW480. We found that SW620 cells are more sensitive than SW480 cells at doses greater than 6 Gy for X­ray or 3 Gy for carbon­ion (C­ion) radiation. Fra­1 expression tended to be decreased by the radiation in a dose­dependent manner in both cell lines; of note, a greater reduction of Fra­1 expression was observed in SW620 cells, especially at 6 Gy of X­ray or 3 Gy of C­ion irradiation, than in SW480 cells, indicating a possible association between Fra­1 downregulation and cellular radiosensitivity. Knockdown of Fra­1 in SW480 cells significantly increased the radiosensitivity to X­ray or C­ion radiation. On the other hand, overexpression of Fra­1 in SW620 cells significantly enhanced the radioresistance to C­ion radiation, suggesting a role of Fra­1 in radioresistance. Furthermore, we found that downregulation of Fra­1 protein in irradiated SW620 cells was regulated via protein degradation through a proteasome­dependent pathway. Overall, our results indicate a role of Fra­1 in radioresistance to both X­ray and C­ion radiation for colorectal cancer cell lines.

Strong radioprotective FGF1 signaling down-regulates proliferative and metastatic capabilities of the angiosarcoma cell line, ISOS-1, through the dual inhibition of EGFR and VEGFR pathways.

BACKGROUND AND PURPOSE: Angiosarcoma is associated with a poor prognosis and is treated with radiotherapy. Although FGF1 is a potential radioprotector, the influence of FGF1 on the malignancy of angiosarcoma remains unknown. MATERIALS AND METHODS: Highly stable FGF1 mutants, which exhibit stronger mitogenic activity than wild-type FGF1, were examined as strong radioprotectors and signaling agonists to clarify the effects of FGF1 on the murine angiosarcoma cell line ISOS-1. RESULTS: FGF1 mutants reduced colony formation by and the in vitro invasion and migration of ISOS-1 cells, in addition to an increase in radiosensitivity to X-rays. In contrast, an FGFR inhibitor blocked the inhibitory effects of FGF1 mutants on colony formation, invasion, and migration. siRNA targeting the Fgfr1 gene showed that strong FGFR1 signaling reduced colony formation by ISOS-1 cells. However, the FGF1 mutant reduced the activation of VEGFRs and EGFRs in ISOS-1 cells more strongly than wild-type FGF1. Moreover, the inhibition of VEGFRs and EGFRs synergistically reduced colony formation by and invasion and migration of ISOS-1 cells. CONCLUSION: These results suggest that strong FGF1 signaling exerts not only radioprotective effects, but also inhibitory effects on proliferative and metastatic capacities of angiosarcoma through the dual inhibition of EGFR and VEGFR pathways.

Genetic Variants in CD44 and MAT1A Confer Susceptibility to Acute Skin Reaction in Breast Cancer Patients Undergoing Radiation Therapy.

PURPOSE: Heterogeneity in radiation therapy (RT)-induced normal tissue toxicity is observed in 10% of cancer patients, limiting the therapeutic outcomes. In addition to treatment-related factors, normal tissue adverse reactions also manifest from genetic alterations in distinct pathways majorly involving DNA damage-repair genes, inflammatory cytokine genes, cell cycle regulation, and antioxidant response. Therefore, the common sequence variants in these radioresponsive genes might modify the severity of normal tissue toxicity, and the identification of the same could have clinical relevance as a predictive biomarker. METHODS AND MATERIALS: The present study was conducted in a cohort of patients with breast cancer to evaluate the possible associations between genetic variants in radioresponsive genes described previously and the risk of developing RT-induced acute skin adverse reactions. We tested 22 genetic variants reported in 18 genes (ie, NFE2L2, OGG1, NEIL3, RAD17, PTTG1, REV3L, ALAD, CD44, RAD9A, TGFßR3, MAD2L2, MAP3K7, MAT1A, RPS6KB2, ZNF830, SH3GL1, BAX, and XRCC1) using TaqMan assay-based real-time polymerase chain reaction. At the end of RT, the severity of skin damage was scored, and the subjects were dichotomized as nonoverresponders (Radiation Therapy Oncology Group grade <2) and overresponders (Radiation Therapy Oncology Group grade ≥2) for analysis. RESULTS: Of the 22 single nucleotide polymorphisms studied, the rs8193 polymorphism lying in the micro-RNA binding site of 3'-UTR of CD44 was significantly (P=.0270) associated with RT-induced adverse skin reactions. Generalized multifactor dimensionality reduction analysis showed significant (P=.0107) gene-gene interactions between MAT1A and CD44. Furthermore, an increase in the total number of risk alleles was associated with increasing occurrence of overresponses (P=.0302). CONCLUSIONS: The genetic polymorphisms in radioresponsive genes act as genetic modifiers of acute normal tissue toxicity outcomes after RT by acting individually (rs8193), by gene-gene interactions (MAT1A and CD44), and/or by the additive effects of risk alleles.

Effects of carbon ion irradiation and X-ray irradiation on the ubiquitylated protein accumulation.

C-ion radiotherapy is associated with improved local control and survival in several types of tumors. Although C-ion irradiation is widely reported to effectively induce DNA damage in tumor cells, the effects of irradiation on proteins, such as protein stability or degradation in response to radiation stress, remain unknown. We aimed to compare the effects of C-ion and X-ray irradiation focusing on the cellular accumulation of ubiquitylated proteins. Cells from two human colorectal cancer cell lines, SW620 and SW480, were subjected to C-ion or X-ray irradiation and determination of ubiquitylated protein levels. High levels of ubiquitylated protein accumulation were observed in the C-ion-irradiated SW620 with a peak at 3 Gy; the accumulation was significantly lower in the X-ray-irradiated SW620 at all doses. Enhanced levels of ubiquitylated proteins were also detected in C-ion or X-ray-irradiated SW480, however, those levels were significantly lower than the peak detected in the C-ion-irradiated SW620. The levels of irradiation-induced ubiquitylated proteins decreased in a time-dependent manner, suggesting that the proteins were eliminated after irradiation. The treatment of C-ion-irradiated SW620 with a proteasome inhibitor (epoxomicin) enhanced the cell killing activity. The accumulated ubiquitylated proteins were co-localized with γ-H2AX, and with TP53BP1, in C-ion-irradiated SW620, indicating C-ion-induced ubiquitylated proteins may have some functions in the DNA repair system. Overall, we showed C-ion irradiation strongly induces the accumulation of ubiquitylated proteins in SW620. These characteristics may play a role in improving the therapeutic ratio of C-ion beams; blocking the clearance of ubiquitylated proteins may enhance sensitivity to C-ion radiation.

FGF18 signaling in the hair cycle resting phase determines radioresistance of hair follicles by arresting hair cycling.

PURPOSE: Telogen (resting phase) hair follicles (HFs) are more radioresistant than their anagen (growth phase) counterparts. Fibroblast growth factor (FGF) 18 is strongly expressed in telogen HFs to maintain the telogen phase, whereas several other FGFs exert radioprotective effects; however, the role of FGF18 in the radioresistance of HFs remains unknown. This study focused on clarifying the role of FGF18 in the radioresistance of telogen HFs and its potential as a radioprotector. METHODS AND MATERIALS: BALB/c mice with telogen or plucking-induced anagen HFs were exposed to total body irradiation with γ-rays at 4 to 12 Gy after intraperitoneal treatment with FGF18 or an FGF receptor inhibitor. A time course analysis was performed histologically and hair growth was observed 14 or 15 days after depilation. Skin specimens were analyzed by DNA microarrays and Western blotting. RESULTS: Telogen irradiation at 6 Gy resulted in transient cell growth arrest, leading to successful hair growth, whereas anagen irradiation failed to promote hair growth. Telogen irradiation did not induce apoptosis in HFs or reduce HF stem cells, whereas anagen irradiation induced apoptosis and reduced stem cell numbers. The Inhibition of FGF receptor signaling during the telogen phase promoted HF cell proliferation; however, hair failed to grow after irradiation. In contrast, recombinant FGF18 induced transient cell growth arrest after anagen irradiation with enhanced DNA repair, leading to the inhibition of apoptosis, maintenance of HF stem cells, and successful hair growth. Moreover, FGF18 reduced the expression levels of genes promoting G2/M transition as well as the protein expression levels of cyclin B1 and cdc2 in skin, and induced G2/M arrest in the keratinocyte cell line HaCaT. CONCLUSIONS: These results suggest that FGF18 signaling mediates radioresistance in telogen HFs by arresting the cell cycle, and that FGF18 has potential as a radioprotector for radiation-induced alopecia.

Cellular internalization of fibroblast growth factor-12 exerts radioprotective effects on intestinal radiation damage independently of FGFR signaling.

PURPOSE: Several fibroblast growth factors (FGFs) were shown to inhibit radiation-induced tissue damage through FGF receptor (FGFR) signaling; however, this signaling was also found to be involved in the pathogenesis of several malignant tumors. In contrast, FGF12 cannot activate any FGFRs. Instead, FGF12 can be internalized readily into cells using 2 cell-penetrating peptide domains (CPP-M, CPP-C). Therefore, this study focused on clarifying the role of FGF12 internalization in protection against radiation-induced intestinal injury. METHODS AND MATERIALS: Each FGF or peptide was administered intraperitoneally to BALB/c mice in the absence of heparin 24 hours before or after total body irradiation with γ rays at 9 to 12 Gy. Several radioprotective effects were examined in the jejunum. RESULTS: Administration of FGF12 after radiation exposure was as effective as pretreatment in significantly promoting intestinal regeneration, proliferation of crypt cells, and epithelial differentiation. Two domains, comprising amino acid residues 80 to 109 and 140 to 169 of FGF12B, were identified as being responsible for the radioprotective activity, so that deletion of both domains from FGF12B resulted in a reduction in activity. Interestingly, these regions included the CPP-M and CPP-C domains, respectively; however, CPP-C by itself did not show an antiapoptotic effect. In addition, FGF1, prototypic FGF, possesses a domain corresponding to CPP-M, whereas it lacks CPP-C, so the fusion of FGF1 with CPP-C (FGF1/CPP-C) enhanced cellular internalization and increased radioprotective activity. However, FGF1/CPP-C reduced in vitro mitogenic activity through FGFRs compared with FGF1, implying that FGFR signaling might not be essential for promoting the radioprotective effect of FGF1/CPP-C. In addition, internalized FGF12 suppressed the activation of p38α after irradiation, resulting in reduced radiation-induced apoptosis. CONCLUSIONS: These findings indicate that FGF12 can protect the intestine against radiation-induced injury through its internalization, independently of FGFRs, suggesting that cellular uptake of FGF12 is an alternative signaling pathway useful for cancer radiation therapy.

Upregulated expression of FGF13/FHF2 mediates resistance to platinum drugs in cervical cancer cells.

Cancer cells often develop drug resistance. In cisplatin-resistant HeLa cisR cells, fibroblast growth factor 13 (FGF13/FHF2) gene and protein expression was strongly upregulated, and intracellular platinum concentrations were kept low. When the FGF13 expression was suppressed, both the cells' resistance to platinum drugs and their ability to keep intracellular platinum low were abolished. Overexpression of FGF13 in parent cells led to greater resistance to cisplatin and reductions in the intracellular platinum concentration. These cisplatin-resistant cells also showed increased resistance to copper. In preoperative cervical cancer biopsy samples from poor prognoses patients after cisplatin chemoradiotherapy, FGF13-positive cells were detected more abundantly than in the biopsy samples from patients with good prognoses. These results suggest that FGF13 plays a pivotal role in mediating resistance to platinum drugs, possibly via a mechanism shared by platinum and copper. Our results point to FGF13 as a novel target and useful prognostic guide for cancer therapy.

Structural stability of human fibroblast growth factor-1 is essential for protective effects against radiation-induced intestinal damage.

PURPOSE: Human fibroblast growth factor-1 (FGF1) has radioprotective effects on the intestine, although its structural instability limits its potential for practical use. Several stable FGF1 mutants were created increasing stability in the order, wild-type FGF1, single mutants (Q40P, S47I, and H93G), Q40P/S47I, and Q40P/S47I/H93G. This study evaluated the contribution of the structural stability of FGF1 to its radioprotective effect. METHODS AND MATERIALS: Each FGF1 mutant was administered intraperitoneally to BALB/c mice in the absence of heparin 24 h before or after total body irradiation (TBI) with γ-rays at 8-12 Gy. Several radioprotective effects were examined in the jejunum. RESULTS: Q40P/S47I/H93G could activate all subtypes of FGF receptors in vitro much more strongly than the wild-type without endogenous or exogenous heparin. Preirradiation treatment with Q40P/S47I/H93G significantly increased crypt survival more than wild-type FGF1 after TBI at 10 or 12 Gy, and postirradiation treatment with Q40P/S47I/H93G was effective in promoting crypt survival after TBI at 10, 11, or 12 Gy. In addition, crypt cell proliferation, crypt depth, and epithelial differentiation were significantly promoted by postirradiation treatment with Q40P/S47I/H93G. The level of stability of FGF1 mutants correlated with their mitogenic activities in vitro in the absence of heparin; however, preirradiation treatment with the mutants increased the crypt number to almost the same level as Q40P/S47I/H93G. When given 24 h after TBI at 10 Gy, all FGF1 mutants increased crypt survival more than wild-type FGF1, and Q40P/S47I/H93G had the strongest mitogenic effects in intestinal epithelial cells after radiation damage. Moreover, Q40P/S47I/H93G prolonged mouse survival after TBI because of the repair of intestinal damage. CONCLUSION: These findings suggest that the structural stability of FGF1 can contribute to the enhancement of protective effects against radiation-induced intestinal damage. Therefore, Q40P/S47I/H93G is pharmacologically one of the most promising candidates for clinical applications for radiation-induced gastrointestinal syndrome.

Sulfation of keratan sulfate proteoglycan reduces radiation-induced apoptosis in human Burkitt's lymphoma cell lines.

This study focuses on clarifying the contribution of sulfation to radiation-induced apoptosis in human Burkitt's lymphoma cell lines, using 3'-phosphoadenosine 5'-phosphosulfate transporters (PAPSTs). Overexpression of PAPST1 or PAPST2 reduced radiation-induced apoptosis in Namalwa cells, whereas the repression of PAPST1 expression enhanced apoptosis. Inhibition of PAPST slightly decreased keratan sulfate (KS) expression, so that depletion of KS significantly increased radiation-induced apoptosis. In addition, the repression of all three N-acetylglucosamine-6-O-sulfotransferases (CHST2, CHST6, and CHST7) increased apoptosis. In contrast, PAPST1 expression promoted the phosphorylation of p38 MAPK and Akt in irradiated Namalwa cells. These findings suggest that 6-O-sulfation of GlcNAc residues in KS reduces radiation-induced apoptosis of human Burkitt's lymphoma cells.

Fibroblast growth factor-12 (FGF12) translocation into intestinal epithelial cells is dependent on a novel cell-penetrating peptide domain: involvement of internalization in the in vivo role of exogenous FGF12.

The extracellular effect of fibroblast growth factor-12 (FGF12) remains unknown because FGF12 cannot activate any fibroblast growth factor receptors (FGFRs), and FGF12 is not currently thought to be released from cells. We reported previously that FGF12 plays an intracellular role in the inhibition of radiation-induced apoptosis. In this study, we demonstrated that recombinant FGF12 was able to be internalized into the cytoplasm of a rat intestinal epithelial cell line, IEC6, and this process was dependent on two novel cell-penetrating peptide (CPP) domains (CPP-M and CPP-C). In particular, CPP-C, composed of ∼10 amino acids, was identified as a specific domain of FGF12 and its subfamily in the C-terminal region (residues 140-149), although CPP-M was a common domain in the internal region of the FGF family. The absence of CPP-C from FGF12 or a mutation (E142L) in the CPP-C domain drastically reduced the internalization of FGF12 into cells. Therefore, CPP-C played an essential role in the internalization of FGF12. In addition, CPP-C was able to deliver other polypeptides into cells as a CPP because an FGF1/CPP-C chimeric protein was internalized into IEC6 cells more efficiently than wild-type FGF1. Finally, intraperitoneally added FGF12 inhibited radiation-induced apoptosis in the intestinal epithelial cells of BALB/c mice, and deletion of the CPP-C domain decreased the inhibition of the apoptosis. These findings suggest that exogenous FGF12 can play a role in tissues by translocating into cells through the plasma membrane, and the availability of this novel CPP provides a new tool for the intracellular delivery of bioactive molecules.

Expression and the role of 3'-phosphoadenosine 5'-phosphosulfate transporters in human colorectal carcinoma.

Sulfation represents an essential modification for various molecules and regulates many biological processes. The sulfation of glycans requires a specific transporter for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) on the Golgi apparatus. This study investigated the expression of PAPS transporter genes in colorectal carcinomas and the significance of Golgi-specific sulfation in the proliferation of colorectal carcinoma cells. The relative amount of PAPST1 transcripts was found to be higher than those of PAPST2 in colorectal cancerous tissues. Immunohistochemically, the enhanced expression of PAPST1 was observed in fibroblasts in the vicinity of invasive cancer cells, whereas the expression of PAPST2 was decreased in the epithelial cells. RNA interference of either of the two PAPS transporter genes reduced the extent of sulfation of cellular proteins and cellular proliferation of DLD-1 human colorectal carcinoma cells. Silencing the PAPS transporter genes reduced fibroblast growth factor signaling in DLD-1 cells. These findings indicate that PAPS transporters play a role in the proliferation of colorectal carcinoma cells themselves and take part in a desmoplastic reaction to support cancer growth by controlling their sulfation status.

Post treatment with an FGF chimeric growth factor enhances epithelial cell proliferation to improve recovery from radiation-induced intestinal damage.

PURPOSE: A fibroblast growth factor (FGF) 1-FGF2 chimera (FGFC) was created previously and showed greater structural stability than FGF1. This chimera was capable of stimulating epithelial cell proliferation much more strongly than FGF1 or FGF2 even without heparin. Therefore FGFC was expected to have greater biologic activity in vivo. This study evaluated and compared the protective activity of FGFC and FGF1 against radiation-induced intestinal injuries. METHODS AND MATERIALS: We administered FGFC and FGF1 intraperitoneally to BALB/c mice 24 h before or after total-body irradiation (TBI). The numbers of surviving crypts were determined 3.5 days after TBI with gamma rays at doses ranging from 8 to 12 Gy. RESULTS: The effect of FGFC was equal to or slightly superior to FGF1 with heparin. However, FGFC was significantly more effective in promoting crypt survival than FGF1 (p < 0.01) when 10 µg of each FGF was administered without heparin before irradiation. In addition, FGFC was significantly more effective at promoting crypt survival (p < 0.05) than FGF1 even when administered without heparin at 24 h after TBI at 10, 11, or 12 Gy. We found that FGFC post treatment significantly promoted 5-bromo-2'-deoxyuridine incorporation into crypts and increased crypt depth, resulting in more epithelial differentiation. However, the number of apoptotic cells in FGFC-treated mice decreased to almost the same level as that in FGF1-treated mice. CONCLUSIONS: These findings suggest that FGFC strongly enhanced radioprotection with the induction of epithelial proliferation without exogenous heparin after irradiation and is useful in clinical applications for both the prevention and post treatment of radiation injuries.

Comparison of expression profiles of several fibroblast growth factor receptors in the mouse jejunum: suggestive evidence for a differential radioprotective effect among major FGF family members and the potency of FGF1.

Several members of the fibroblast growth factor (FGF) family have the potential to protect the intestine against the side effects of radiation therapy. FGF1 is capable of signaling through all subtypes of FGF receptors (FGFRs), whereas FGF7 and FGF10 activate only the epithelial-specific subtype, FGFR2IIIb (FGFR2b). The present study compared the protective activity of FGF1, FGF7 and FGF10 and examined the profiles of FGFR expression in the jejunum of BALB/c mice given total-body irradiation (TBI) with gamma rays. TBI caused drastic increases in FGFR1-4 transcript levels in the jejunum. However, FGFR2b protein temporarily decreased at 12 and 24 h after irradiation. FGF1 pretreatment minimized the number of apoptotic cells in jejunal crypts at 16 and 24 h after irradiation and increased crypt survival most effectively. In addition, pretreatment with FGF7 or FGF10 decreased FGFR1 transcript levels. The greater effectiveness of FGF1 to enhance crypt survival was also observed even when each FGF was administered 1 h after irradiation. These findings indicate that FGF1 is more potent than FGF7 or FGF10 for protection of the intestine against radiation exposure and suggest that the profiles of FGFR expression in the intestine favor the FGF1 signaling pathway before and during the initial period after irradiation.

Evaluation of radiation-induced hair follicle apoptosis in mice and the preventive effects of fibroblast growth factor-1.

Radiation-induced hair loss is a clinically important, but under-researched topic. The aim of the study was to develop an in vivo assay system for radiation-induced apoptosis in hair follicles to promote hair research and exploit new radioprotectors. BALB/c mice received total body irradiation (TBI) with gamma-rays at doses in the range from 8 to 16 Gy at 6 days after depilation. Pathological changes were detected progressively in the hair follicles over the time course after TBI and the dystrophy was evaluated on the basis of stage-specific parameters reported previously, which were found to be well-suited for classification of the radiation-induced hair follicle dystrophy. As a result, regression from anagen to catagen was determined in these follicles after irradiation. In addition, radiation-induced apoptosis was a good early dystrophic parameter. In this system, it was found that fibroblast growth factor-1 effectively prevented hair follicle apoptosis in mice.