Multiple Lymphaticovenular Anastomoses for Chyluria in Klippel-Trenaunay Syndrome.

ABSTRACT: Klippel-Trenaunay syndrome (KTS) is characterized by port-wine stains, mixed vascular malformations, and soft tissue and bone hypertrophy. Klippel-Trenaunay syndrome is occasionally complicated by chyluria, for which there is no effective treatment currently. We report a case of KTS complicated by intractable chyluria and hematuria due to a lymphatic-ureteral fistula. The patient was successfully treated with multiple lymphaticovenular anastomoses (LVAs).A 66-year-old woman with an enlarged left lower extremity since childhood was diagnosed with KTS. At 60 years of age, she developed chyluria (urine albumin, 2224 µg/mL) and hematuria. Lymphoscintigraphy showed a lymphatic-ureteral fistula near the ureterovesical junction. Conservative treatment was ineffective. She also developed left lower extremity lymphedema, which gradually worsened. Leg cellulitis and purulent pericarditis developed because of hypoalbuminemia (minimum serum albumin level, 1.3 g/dL).We performed 14 LVAs in 2 surgeries to reduce lymphatic fluid flow through the lymphatic-ureteral fistula. The chyluria and hematuria resolved soon after the second operation, and the urine albumin level decreased (3 µg/mL). After 28 months, she had no chyluria or hematuria recurrence and her serum albumin level improved (3.9 g/dL). Multiple LVAs can definitively treat chyluria caused by a lymphatic-ureteral fistula in patients with KTS.

Videofluorographic Analysis of Swallowing Function after Total Glossolaryngectomy.

SUMMARY: Surgeons have traditionally believed that swallowing is mainly dependent on gravity after total glossolaryngectomy. However, swallowing function after total glossolaryngectomy varies widely among patients, and a thorough analysis is lacking. The authors aimed to clarify the swallowing function after total glossolaryngectomy and determine whether it is primarily dependent on gravity. The authors retrospectively analyzed videofluorographic examinations of patients who underwent total glossolaryngectomy and free or pedicle flap reconstruction. The authors enrolled 20 patients (12 male; mean age, 61 years; age range, 43 to 89 years). All patients demonstrated constriction of the reconstructed pharynx to some degree, and no patient's ability to swallow was dependent on gravity alone. Videofluorography showed excellent barium clearance in eight patients and poor clearance in 12. All patients with excellent clearance showed strong constriction of the posterior pharyngeal wall, whereas only 8.3 percent of the patients with poor clearance showed adequate constriction, which was significantly different ( p = 0.0007). Velopharyngeal closure and lip closure also contributed significantly to excellent clearance ( p = 0.041). The shape of the reconstructed pharynx (depressed, flat, protuberant) showed no statistically significant association with excellent clearance. Contrary to previous understanding, constriction of the remnant posterior pharyngeal wall played an important role in swallowing after total glossolaryngectomy, and gravity played a secondary role. Dynamic posterior pharyngeal wall movement might result from the increased power of the pharyngeal constrictor muscle and compensate for the immobility of the transferred flap. A well-functioning pharyngeal constrictor muscle and complete velopharyngeal and lip closures can contribute to excellent barium clearance in patients after total glossolaryngectomy. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

A Double-Barreled Fibular Graft for the Reconstruction of Both Forearm Bones and Humeroradial Joint after a Tumor Resection.

A double-barreled fibular graft was used to reconstruct both forearm bones and the humeroradial joint after tumor resection. The patient had a tumor of radius that invaded the ulna and extensor groups. After a wide tumor resection, vascularized fibular autograft and soft tissue reconstruction was performed. A fibular graft were placed as a double barrel in the proximal ulnar and radial defects including the radial head and fixed using two locking plates. Simultaneously, reconstruction of the humeroradial joint and wrist dorsiflexion was performed. Two years postoperatively, the patient is satisfied with his elbow function while performing activities of daily living. Although amputation was one of the options considered during the preoperative planning in this case, the affected limb could be preserved by grafting a double-barreled fibula and tendon transfer, which could maintain the function of his upper left limb.

Successful Management of a Severe Sacral Pressure Injury Penetrating to the Retroperitoneum.

INTRODUCTION: Reports of retroperitoneal infection related to a sacral pressure injury (PI) are rare, and none of the reports described the direct spread of infection through the sacrum to the retroperitoneum. The authors present, to their knowledge, the first report of a severely infected PI that showed full-thickness sacral destruction and direct retroperitoneal penetration. CASE REPORT: A 63-year-old female was referred for management of a stage 4 sacral PI complicated by a retroperitoneal abscess. The patient's comorbidities were diabetes mellitus and pemphigus foliaceus with steroid therapy-induced immunosuppression. Upon admission, the patient presented with a sacral PI producing copious purulent discharge that measured 5 cm × 3 cm. Magnetic resonance imaging revealed full-thickness sacral bone destruction and a massive retroperitoneal abscess, suggesting the sacral PI directly penetrated to the retroperitoneal space. Antibiotics were administered, and surgical debridement and sequestrectomy were performed. Negative pressure wound therapy (NPWT) with continuous saline irrigation was initiated. The patient's mesorectum was exposed within the retroperitoneal space. Therefore, a nonadhesive wound dressing was applied before placing the irrigation tube to avoid perforating the rectum. Because the patient had fragile skin secondary to long-standing pemphigus foliaceus and steroid treatment, a liquid skin protectant and hydrocolloid wound dressing were applied. The infection was successfully controlled with NPWT with saline irrigation. The patient experienced no rectal injury or skin rupture, and surgical closure was performed after 75 days. Although partial wound dehiscence occurred because of the poor condition of the skin, the resultant open wound was managed conservatively. The patient showed no retroperitoneal abscess recurrence 6 months later. CONCLUSIONS: A rare case of an intractable sacral PI complicated by retroperitoneal abscess was successfully managed in an immunocompromised patient. Notably, NPWT with saline irrigation was useful in controlling the patient's severe retroperitoneal infection.

Lymphaticovenular anastomosis for lymph vessel injury in the pelvis and groin.

BACKGROUND: Lymphatic diseases due to lymph vessel injuries in the pelvis and groin require immediate clinical attention when conventional treatments fail. We aimed to clarify the effectiveness of and indications for lymphaticovenular anastomosis (LVA) to treat these lymphatic diseases. METHODS: We retrospectively evaluated six patients who underwent LVA for lymphatic diseases due to lymph vessel injuries in the pelvis and groin. Specific pathologies included groin lymphorrhea (N = 3), chylous ascites (N = 2), and retroperitoneal lymphocele (N = 1). The maximum lymphatic fluid leakage volume was 150-2600 mL daily. Conventional treatments (compression, drainage, fasting, somatostatin administration, negative pressure wound therapy, or lymph vessel ligation) had failed to control leakage in all cases. We performed lower extremity LVAs after confirming the site of lymph vessel injury using lymphoscintigraphy. We preferentially placed LVAs in thigh sites that showed a linear pattern by indocyanine green lymphography. Postoperative lymphatic fluid leakage volume reduction was evaluated, and leakage cessation was recorded when the drainage volume approached 0 mL. RESULTS: LVA was performed at an average of 4.3 sites (range, 3-6 sites) in the thigh and 2.7 sites (range, 0-6 sites) in the lower leg. Lymphatic fluid leakage ceased in all cases after a mean of 6 days (range, 1-11 days) postoperatively. No recurrence of symptoms was observed during an average follow-up of 2.9 (range, 0.5-5.5) years. CONCLUSIONS: LVA demonstrates excellent and rapid effects. We recommend lower extremity LVA for the treatment of lymphatic diseases due to lymph vessel injuries in the pelvis and groin.

Securing split-thickness skin grafts using negative-pressure wound therapy without suture fixation.

OBJECTIVE: Negative-pressure wound therapy (NPWT) is generally applied as a bolster for split-thickness skin grafts (STSG) after the graft has been secured with sutures or skin staples. In this study, NPWT was applied to secure STSGs without any sutures or staples. Surgical outcomes of using NPWT without sutures was compared with a control group. METHODS: Patients with STSGs were divided into two groups: a 'no suture' group using only NPWT, and a control group using conventional fixings. In the no suture group, the grafts were covered with meshed wound dressing and ointment. The NPWT foam was placed over the STSG and negative pressure applied. In the control group, grafts were fixed in place using tie-over bolster, securing with fibrin glue, or NPWT after sutures. RESULTS: A total of 30 patients with 35 graft sites participated in the study. The mean rate of graft take in the no suture group was 95.1%, compared with 93.3% in the control group, with no significant difference between them. No graft shearing occurred in the no suture group. Although the difference did not reach statistical significance, mean surgical time in the no suture group (31.5 minutes) tended to be shorter than that in the control group (55.7 minutes). CONCLUSION: By eliminating sutures, the operation time tended to be shorter, suturing was avoided and suture removal was not required meaning that patients could avoid the pain associated with this procedure. Furthermore, the potential for staple retention and its associated complications was avoided, making this method potentially beneficial for both medical staff and patients.

Large-defect Resurfacing: A Comparison of Skin Graft Results Following Sarcoma Resection and Traumatic Injury Repair.

INTRODUCTION: Soft tissue sarcomas are rare neoplasms, and most plastic surgeons do not commonly resurface large tissue defects after a wide resection of these tumors. OBJECTIVE: The purpose of this study is to elucidate the clinical results of large skin grafts after wide sarcoma resection by comparison with grafts for traumatic skin defects. MATERIALS AND METHODS: A retrospective review was performed of patients who received skin grafts > 50 cm2 after traumatic injury or wide sarcoma resection from 2014 to 2016. Patient medical records were reviewed; graft take rate, graft loss, and days to complete epithelialization were compared between the 2 groups. RESULTS: In the sarcoma group (n = 8), 5 grafts were partially lost; the sarcoma group mean graft take rate of 67.5% ± 30.0% was significantly lower than that of the trauma group (n = 7) at 99.6% ± 1.1%. The mean time to complete epithelialization from the skin graft placement in the sarcoma group was 113.3 ± 66.0 days, which was significantly longer than that of the trauma group (40.3 ± 38.0 days). Wounds located around the shoulder joint in 2 sarcoma group patients did not heal even after 300 days of conservative treatment; 1 required a secondary flap. CONCLUSIONS: The results of skin grafting for resurfacing large defects after sarcoma resection are inferior to those for traumatic injury repair. Skin grafts may fail because the blood supply for the wound bed is impaired during resection. Furthermore, due to the wound bed movement, epithelialization over muscles of the shoulder joint is difficult to achieve, and skin grafts in this region will likely fail.

Toxic Shock Syndrome Caused by Staphylococcal Infection After Breast Implant Surgery: A Case Report and Literature Review.

Toxic shock syndrome is a rare but life-threatening complication after breast implant surgery. We describe a 77-year-old woman who developed toxic shock syndrome caused by methicillin-resistant Staphylococcus aureus after breast implant reconstruction. Despite a high fever and markedly increased white blood cell count, suggesting severe infection, she initially had no symptoms of local findings, such as wound swelling and redness of the breast. Soon after diagnosis of toxic shock syndrome and removal of her breast implant, she was recovered from the shock state. To date, 16 cases of toxic shock syndrome have been reported, including this case, and they were related to breast implants or tissue expander surgery. The common and noteworthy characteristic of these cases was the lack of local findings, such as swelling or redness, which suggests infection. Therefore, early diagnosis is generally difficult, and the initiation of proper treatment can be delayed without knowledge of this characteristic. Toxic shock syndrome requires early diagnosis and treatment. If the patient has a deteriorated vital sign after breast implant surgery or tissue expander breast reconstruction, toxic shock syndrome should be suspected, even if there are no local signs of infection, and removal of the artifact should be considered as soon as possible.

Pharyngeal reconstruction by anterolateral thigh flap with vastus lateralis muscle transfer for effective swallowing after total glossolaryngectomy: A case report.

BACKGROUND: We report a novel reconstruction technique that maintained effective swallowing after total glossolaryngectomy (TGL) by restoring pharyngeal constriction using a vascularized vastus lateralis muscle transfer. METHODS: A 65-year-old male with recurrent tongue cancer underwent TGL and anterolateral thigh flap reconstruction with the vastus lateralis muscle. The bilateral cut ends of the remaining posterior pharyngeal wall constrictor muscle were sutured to the transferred vastus lateralis muscle so that the two muscles encircled the reconstructed pharynx. The femoral nerve of the vastus lateralis muscle was coapted to the hypoglossal nerve. RESULTS: Videofluorographic examination showed the contrast bolus flowing smoothly with little assistance from gravity. Laryngoscopic examination showed circumferential constriction of the reconstructed pharynx. The patient could swallow soft food without placing the bolus in his posterior oral cavity or drinking simultaneously. CONCLUSION: The restoration of pharyngeal constriction introduces the possibility of functional swallowing in patients after TGL.

Simultaneous deep inferior epigastric and bilateral anterolateral thigh perforator flap reconstruction of an extended perineoscrotal defect in Fournier's gangrene: A case report.

Fournier's gangrene is lethal necrotizing fasciitis that involves the perineum and external genitalia. We describe the case of a 52-year-old man with Fournier's gangrene who underwent reconstruction of an extensive perineoscrotal defect using three pedicled perforator flaps. Three debridement procedures resulted in a skin and soft tissue defect of 36 × 18 cm involving the perineum, scrotum, groin, medial thigh, buttocks, and circumferential perianal area and left the perforating arteries originating from these locations unavailable for reconstruction. We repaired the defect using left deep inferior epigastric artery perforator (DIEP) (29 × 8 cm) and bilateral anterolateral thigh perforator (ALT) flaps (35 × 8 cm and 22 × 7 cm). The flaps reached the defect without tension, and the defect was successfully covered without a skin graft. No postoperative complications occurred except for epidermal necrosis involving a tiny part of the DIEP flap tip. Nine months postoperatively, the patient experienced no impairment of bowel function or hip joint movement. There was also no avulsion or ulceration of the reconstructed perineal skin, and the cosmetic appearances of the healed wound and donor site were satisfactory. The combination of these three perforator flaps enabled us to achieve a satisfactory outcome while avoiding skin grafts.

Anatomic Reconstruction of Lateral Ankle Ligaments and Both Peroneus Tendons After Open Fracture Dislocation of the Ankle: A Case Report.

Extensive soft tissue defects of the ankle are an uncommon but challenging problem that require a combination of reconstructive options. We report the case of a complex injury involving the skin, lateral ankle ligaments, and peroneal tendons that were anatomically reconstructed. A 15-year-old girl was injured in an automobile accident resulting in extensive soft tissue defects and marked instability of her right ankle. The lower two-thirds of the anterior talofibular ligament (ATFL) had segmental defects, and calcaneofibular ligament (CFL) was completely torn, and both peroneal longus and brevis tendons were severely damaged. Initial debridement was performed on the day on injury. Two weeks after injury, the ATFL and CFL were reconstructed using a semitendinosus autograft and suture tape augmentation. Both peroneal tendons were reconstructed using a gracilis autograft. The skin defect (10 × 10 cm) was covered with an anterolateral thigh flap. After removing a short leg cast at 3 weeks postoperatively, the patient started range of motion exercises without using any brace. Weightbearing was allowed at 4 weeks. At the 24-month follow-up examination, she had returned to her preoperative level of work and sports activities.

Streptococcus pyogenes-purpura fulminans as an invasive form of group A streptococcal infection.

BACKGROUND: Streptococcus pyogenes is an uncommon pathogen of purpura fulminans, and the pathogenesis of S. pyogenes-purpura fulminans remains unclear because of paucity of cases. We reported a pediatric case of S. pyogenes-purpura fulminans with literature review of the disease. CASE PRESENTATION: A 3-year-old boy showed limping, lethargy and acral gangrene within 24 h. A diagnosis of S. pyogenes-purpura fulminans was made for bacterial isolation from throat and peripheral blood. Intensive therapy led to a survival with amputation of the left distal metatarsal bone, and normal development. The isolated M12 carried no mutation of csrS/R or rgg. Thrombophilia or immunodeficiency was excluded. DISCUSSION: Twelve-reported cases (9 pediatric and 3 elderly) of S. pyogenes-purpura fulminans started with shock and coagulopathy. Five patients age < 8 years had no underlying disease and survived. One youngest and two immunocompromised patients died. CONCLUSION: Streptococcus pyogenes-acute infectious purpura fulminans is a distinctive rare form of aggressive GAS infections.

Successful treatment of severe facial lymphedema by lymphovenous anastomosis.

BACKGROUND: Facial edema is a common complication after neck dissection and/or chemoradiotherapy for head and neck cancer. Edema subsides spontaneously in most cases but sometimes persists, in which case surgical intervention is required. We report a case of severe facial edema that showed significant improvement upon lymphovenous anastomosis (LVA). METHODS: A 66-year-old man with oral floor cancer developed progressive facial lymphedema after tumor resection, bilateral neck dissections, chemoradiotherapy, and fibular and rectus abdominis musculocutaneous flap transfer. His eyesight was completely disturbed due to severe eyelid edema. The LVAs were performed in the bilateral preauricular area. Surgical findings showed stagnation of the lymphatic fluids in dilated lymphatic vessels, which were drained to the superficial temporal veins by LVA. RESULTS: The edema subsided rapidly and the patient's eyesight returned as soon as 4 days postoperatively. CONCLUSION: Using LVA in the preauricular region can be a choice of surgical treatment for severe facial edema.

Non-anatomical reconstruction of lateral ulnar collateral ligament of the elbow after tumor resection.

We present the case of an 80-year-old man with a tumor recurrence on his right arm 6 years after initial treatment. The lateral aspect of the elbow joint, involving overlaying skin, muscles, tendons, joint capsule, lateral collateral ligament complex, the lateral 1/3 of the capitellum, and lateral epicondyle of humerus were excised in the tumor resection. Intraoperative assessment revealed multidirectional instability of the elbow, and joint stabilization was needed. Because the lateral epicondyle was resected, graft placement in an anatomical position was impossible to carry out. Therefore, non-anatomical reconstruction of lateral ulnar collateral ligament with palmaris longus tendon graft was performed. The skin was reconstructed using an antegrade pedicled radial forearm flap. For wrist extension reconstruction, the pronator quadratus tendon was transferred to the extensor carpi radialis brevis tendon. One year after the operation, elbow range of motion was 5-130°. The patient remains symptom free. The Mayo elbow performance score is good. The Musculoskeletal Tumor Society rating score is excellent. To our knowledge, this is the first report of an elbow lateral ulnar collateral ligament reconstruction after tumor resection.

The mechanism of cross-resistance to proteasome inhibitor bortezomib and overcoming resistance in Ewing's family tumor cells.

EWS-Fli1 plays important roles in oncogenesis of Ewing's family tumors (EFTs). We have reported that EWS-Fli1 inhibits p21(waf1/cip1) and p27(kip1) expressions, which are degraded by the ubiquitin-proteasome pathway. Bortezomib efficiently up-regulated p21(waf1/cip1) and p27(kip1) expression, and induced apoptosis accompanied by the expression of cleaved-PARP, DR4 and activated caspase-8 in EFT cells. Since most EFTs deaths result from the tumor being resistant to chemotherapeutic drugs, the effects of novel anti-tumor reagents on drug-resistant tumors were next investigated. The results demonstrated that the drug-resistant EFT clones were cross-resistant to bortezomib probably due to the over-expression of the efflux pumps, P-glycoprotein and MRP1. We further investigated whether the efflux pump inhibitors would modulate the effects of bortezomib. The combination of P-gp-specific or MRP1-specific inhibitors could enhance the anti-tumor effects of bortezomib on the drug-resistant clones. These data suggest that bortezomib might be a substrate of P-gp and MRP1. Although bortezomib would be effective on the primary EFTs, it is necessary to pay attention to the resistance to bortezomib in clinical trials for the advanced cases. The combination of bortezomib and the efflux pump inhibitors might be a promising method as a novel molecular target therapy for advanced EFTs.

The effects of histone deacetylase inhibitors on the induction of differentiation in chondrosarcoma cells.

PURPOSE: Histologically, chondrosarcomas represent the degree of chondrogenic differentiation, which is associated with the prognosis of the disease. Histone acetylation and deacetylation play key roles in the regulation of chondrocytic differentiation. Here, we describe the antitumor effects of histone deacetylase (HDAC) inhibitors as differentiating reagents on chondrosarcomas. EXPERIMENTAL DESIGN: We examined the effects of a HDAC inhibitor, depsipeptide, on the growth of chondrosarcoma cell lines. We also investigated the modulation of the expression levels of extracellular matrix genes and the induction of phenotypic change in chondrosarcoma cells treated with depsipeptide. Finally, we examined the antitumor effect of depsipeptide on chondrosarcoma in vivo. RESULTS: Depsipeptide inhibited the growth of chondrosarcoma cells by inducing cell cycle arrest and/or apoptosis. HDAC inhibitors increased the expression of the alpha1 chain of type II collagen (COL2A1) gene due to the enhanced histone acetylation in the promoter and enhancer. Depsipeptide also up-regulated the expressions of aggrecan and the alpha2 chain of type XI collagen (COL11A2) mRNA in a dose-dependent manner. Moreover, long-term treatment with a low dose of depsipeptide resulted in the induction of differentiation into hypertrophic phenotype, as shown by the increment of the alpha1 chain of type X collagen (COL10A1) expression in chondrosarcoma cells. In vivo studies and histologic analyses confirmed that depsipeptide significantly inhibited tumor growth and induced differentiation into the hypertrophic and mineralized state in chondrosarcoma cells. CONCLUSIONS: These results strongly suggest that HDAC inhibitors may be promising reagents for use as a differentiating chemotherapy against chondrosarcomas.

The possible role of EWS-Fli1 in evasion of senescence in Ewing family tumors.

The chromosomal translocation t(11;22) yields the EWS-Fli1 fusion gene and is associated with oncogenesis of Ewing family tumors (EFT). In this study, using the RNA interference method, we show that EWS-Fli1-targeting small interfering RNAs (siRNA) depleted EWS-Fli1 protein and caused growth inhibition in EFT cells with the accumulation of p27 protein and the down-regulation of Skp2 protein in dose-dependent, time-dependent, and sequence-specific manners. Depletion of EWS-Fli1 subacutely elicited a senescence-like phenotype, but not apoptosis, in EFT cells. Furthermore, not only the knockdown of p27, but also the forced expression of Skp2, reduced the expression levels of p27 protein and partially rescued senescence-like phenotype caused by EWS-Fli1-targeting siRNAs. The accumulation of p27 protein in EWS-Fli1-depleted cells inhibited cdk2 kinase activity and was related to the stability of p27 protein, which resulted from a decrease in Skp2 protein. Immunohistochemical analysis of p27 and Skp2 proteins in EFT samples revealed that there was an inverse relationship between the expression profiles of p27 and Skp2 proteins. These findings indicate an important role of EWS-Fli1 in the prevention of senescence, leading to the unlimited growth and oncogenesis of EFT cells through a decrease in the stability of p27 protein due to increased action of Skp2-mediated 26S proteasome degradation.

Involvement of P-glycoprotein and MRP1 in resistance to cyclic tetrapeptide subfamily of histone deacetylase inhibitors in the drug-resistant osteosarcoma and Ewing's sarcoma cells.

Despite recent improvements in multimodal therapies for osteosarcoma (OS) and Ewing's family of tumors (EFTs), the prognosis of relapsed cases remains very poor because of the resistance to chemotherapy. Histone deacetylase inhibitors (HDACIs), including members of the cyclic tetrapeptide family such as FK228 and apicidin, are novel antitumor agents that can induce cell cycle arrest and apoptosis in various cancer cells. HDACIs also exhibit potent antitumor effects on OS and EFTs. However, to date there have been no studies to our knowledge reporting the effects of HDACIs on drug-resistant OS and EFTs. Here, we demonstrated that FK228 and apicidin exhibited strong resistance in doxorubicin-resistant clones of OS and EFTs expressing P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) and that P-gp and MRP1 might play a crucial role in the resistance mechanism to FK228 and apicidin. A P-gp inhibitor (verapamil) and an MRP1 inhibitor (MK571) could independently reverse the resistance to FK228 and apicidin in the drug-resistant clones. Moreover, the combination of verapamil and MK571 could enhance HDACI-induced cell number reduction in drug-resistant clones to a similar extent as that in their parental clones. Although these findings suggest the difficulty in treating drug-resistant tumors expressing P-gp and/or MRP1 with these HDACIs, the combination of P-gp and MRP1 inhibitors might reverse the resistance to the HDACIs in the treatment of those tumors. Because HDACIs are potent and promising antitumor drugs and seem to be close to clinical use, it is necessary to pay attention to the resistance mechanisms against HDACIs.

Focal adhesion kinase is activated in invading fibrosarcoma cells and regulates metastasis.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is overexpressed in several human cancers, and induces survival, proliferation and motility of cells in culture. Phosphorylation of FAK has been studied extensively in vitro, but little is known about its regulation during tumor invasion in vivo. In the current study, green fluorescent protein (GFP) was expressed stably in an invasive murine fibrosarcoma cell line for the purpose of discrimination between tumor and normal cells. Under fluorescence microscopy, the tumor was highly fluorescent, and the margin between the tumor and normal tissue was clearly demarcated. Using this invasion model, we showed localization of pY397-FAK expression in the infiltrative edge of tumors. We reproduced local invasion in vivo using a tumor tissue culture method in a three dimensional collagen gel. Phosphorylation of FAK is also upregulated in invading fibrosarcoma cells under in vitro conditions. Expression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) in 2,472 cells decreased FAK phosphorylation without changing total FAK levels. FRNK inhibited the motility of 2,472 cells, and reduced invasion in vitro. Although FRNK did not affect cell growth, it inhibited experimental metastases in syngenic mice. These results demonstrate that the phosphorylation of FAK might be specifically upregulated in invading fibrosarcoma cells and regulate their invasion and metastasis.

Transactivation of cyclin E gene by EWS-Fli1 and antitumor effects of cyclin dependent kinase inhibitor on Ewing's family tumor cells.

Chromosomal translocation t(11; 22)(q24; q12) is detected in approximately 90% of Ewing's family tumors (EFTs) including Ewing's sarcoma and primitive neuroectodermal tumor. This results in the formation of the EWS-Fli1 fusion gene, which produces EWS-Fli1 fusion protein. This chimerical gene product acts as an aberrant transcriptional activator, which may be responsible for the tumorigenesis of EFTs. We have previously reported that cyclin E expression was upregulated in EFT cells and in EWS-Fli1 transformed fibroblastic cells. However, the mechanism of the overexpression of cyclin E by EWS-Fli1 is still unknown. In our study, we investigated the mechanism of transactivation of the cyclin E gene in EFT cells. We found that EWS-Fli1 enhanced the activity of the cyclin E gene promoter partially through E2F binding sites in the promoter. In addition, the basic transcriptional factor, Sp1, might also be involved in the transactivation of the cyclin E gene by EWS-Fli1. To study the biological significance of cyclin E overexpression in EFT cells, we used flavopiridol, a pan-cyclin-dependent kinase (CDK) inhibitor and found that flavopiridol efficiently suppressed the growth of EFT cells in vitro and in vivo by the inhibition of cyclinE/CDK2 kinase activity and the induction of apoptosis. These results suggest that targeting of the cyclin/CDK complex may provide new insight into treatment of EFTs.