[Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma].

OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION: Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.

Nuclear expression of MDM2 in hibernoma: a potential diagnostic pitfall.

Hibernoma is a rare benign adipocytic tumor composed of a proliferation of brown and white fat cells varying in their proportions. The tumor may also contain fat cells resembling lipoblasts, which makes it difficult to distinguish it from atypical lipomatous tumor/well differentiated liposarcoma (ALT/WDLS). Although nuclear expressions of murine double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) are widely used as immunohistochemical surrogate markers for ALT/WDLS, the utility of these proteins in distinguishing between hibernoma and ALT/WDLS still remains to be elucidated. We evaluated immunohistochemical expressions of MDM2 and CDK4 in 10 hibernomas expressing uncoupling protein-1 (UCP-1), a mitochondrial protein transporter consistently expressed in brown fat cells, and lacking MDM2 gene amplification, which was analyzed by fluorescence in situ hybridization (FISH). In contrast to the data previously obtained, nuclear expression of MDM2 was observed in 100% (10/10 cases) of the hibernomas irrespective of the proportion of brown fat cells, whereas no cases were positive for CDK4. The tumors also showed almost concurrent expression of p53 (in 9/10 cases) and ubiquitin-specific-processing protease 7 (USP7) (in 10/10 cases), which deubiquitinates and stabilizes MDM2, potentially resulting in its nuclear expression without MDM2 gene amplification. MDM2 expression may thus be a diagnostic pitfall for hibernoma particularly in differentiating it from ALT/WDLS.

The Utility of MDM2 and CDK4 Immunohistochemistry and MDM2 FISH in Craniofacial Osteosarcoma.

Craniofacial osteosarcoma is rare (2-10% of all osteosarcomas). Most low grade fibroblastic osteosarcomas of the long bones are characterized by amplification of chromosome12q including MDM2 and CDK4 genes. This study aims to investigate the utility of MDM2 and CDK4 immunostains as well as MDM2 FISH in craniofacial osteosarcomas as a means of distinguishing them from benign fibro-osseous lesions. Cases of primary osteosarcoma and benign fibro-osseous lesions of the craniofacial bones were identified in the diagnostic pathology archives. MDM2 (SMP14 and/or IF2) and CDK4 (D9G3E and/or DCS-31) immunostains were performed on a representative block from each osteosarcoma and benign case. Fluorescence in situ hybridization (FISH) for MDM2 was performed on non-decalcified osteosarcomas. In osteosarcomas, the rate of expression of either MDM2 IF2, MDM2 SMP14, CDK4 DCS-31, or CDK4 D9G3E was 72.7% (8/11 cases), usually focal and weak. Using the MDM2 IF2 clone and the CDK4 DCS-31 clone, MDM2 and CDK4 were negative in lesional cells in all 14 benign fibro-osseous lesions. Using the IF2 and SMP14 clones, MDM2 nuclear expression was present in associated osteoclast-like giant cells in both benign and malignant cases. Of 4 successful cases, 1 high grade osteosarcoma was positive for MDM2 amplification. MDM2 or CDK4 expression or MDM2 amplification may aid in a diagnosis of head and neck osteosarcoma. However, when absent, sarcoma is not excluded. Due to focal weak expression of MDM2 in tumor cells in conjunction with nuclear expression in associated giant cells, caution should be exercised when interpreting positive stains.

Overexpression of cyclin-dependent kinase 4 protein in extramammary Paget's disease.

Cyclin-dependent kinase 4 and 6 (CDK4/6) plays an important role in cell cycle progression, and the CDK4/6-cyclin D1 complex controls the cell cycle transition from G1 phase to S phase. CDK4 is enhanced in several types of cancers and CDK4/6 inhibitors attenuate the proliferation of several types of cancer in vitro/in vivo. The purpose of our study was to investigate the expression pattern of CDK4 and evaluate its clinical importance in extramammary Paget's disease (EMPD). Almost all EMPD tissues were positive for CDK4, and metastatic lesions had a similar immunostaining intensity to primary lesions. In addition, CDK4 protein levels were positively correlated with those of cyclin D1 protein. Taken together, CDK4 may assume a crucial role in EMPD progression.

Lipomas of the Oral Cavity: Utility of MDM2 and CDK4 in Avoiding Overdiagnosis as Atypical Lipomatous Tumor.

Traumatized lipomas with degenerative change may demonstrate histopathologic features that mimic atypical lipomatous tumor (ALT). Previously reported series of ALT involving the oral cavity preceded routine use of MDM2 and CDK4 immunohistochemistry. Our aim is to evaluate MDM2 and CDK4 immunohistochemical expression in adipocytic tumors arising in this site, in conjunction with the histiocytic marker PU.1, to determine whether MDM2 and CDK4 impacts classification. 17 cases originally diagnosed as ALT were retrieved and immunohistochemical studies for MDM2, CDK4 and PU.1 were performed. FISH analysis for MDM2 amplification was performed in select cases. For this study group, the male:female ratio was 9:8 and the median age was 62 (range 41-88). All 17 cases presented as well- or predominantly well-circumscribed proliferations of variably sized, mature adipocytes exhibiting uni- or multi-vacuolation with occasional scalloped nuclei and mild nuclear atypia. Variable amounts of fibrous stroma with focal myxoid change and bland spindle cells were identified in 14/17 cases. Lipoblasts or atypical hyperchromatic stromal cells were not identified in any cases. 14 of 17 cases were negative for MDM2 and CDK4 in tumor cells and 11 of these 14 showed weak nuclear positivity for MDM2 in histiocytes. 3 of 17 cases showed weak, multifocal immunohistochemical expression of MDM2 and CDK4. PU.1 highlighted histiocytes in all 17 cases. FISH analysis for MDM2 amplification was negative in all 3 cases with weak MDM2/CDK4 expression. All cases were reclassified as lipoma with degenerative changes. ALT, in all likelihood, is less common than previously thought in this anatomic location and best diagnosed with ancillary studies. MDM2 expression in histiocytes is best interpreted in conjunction with CDK4 immunohistochemistry and confirmatory FISH for MDM2 amplification.

MiR-1301-3p inhibits human breast cancer cell proliferation by regulating cell cycle progression and apoptosis through directly targeting ICT1.

BACKGROUND: MiRNAs regulate a variety of biological processes, such as cell proliferation and apoptosis and play critical roles in cancer progression. Accumulating studies have demonstrated that miR-1301-3p could regulate the development and progression of multiple cancers, but its biological behaviors in breast cancer (BC) are still elusive. METHODS: The expression of miR-1301-3p was determined in BC tissues and cell lines using quantitative real-time PCR analysis. The effects of miR-1301-3p on BC cell growth, proliferation, cell cycle distribution, and apoptosis were also explored in vitro using MTT, colony formation and Flow cytometry assays. The potential target gene of miR-1301-3p was determined by dual-luciferase reporter assay and verified by quantitative real-time PCR and western blot analysis. RESULTS: We found the expression of miR-1301-3p was observably significantly down-regulated in BC tissues and cell lines. MiR-1301-3p expression in BC tissues was significantly associated with tumor size and clinical stage. Gain-of-function assays demonstrated that miR-1301-3p inhibited the cell growth and proliferation in breast cancer cell lines, MCF-7 and T-47D. Moreover, up-regulation of miR-1301-3p induced cell cycle G0/G1 phase arrest and apoptosis. Mechanistically, up-regulation of miR-1301-3p reduced the expression of CDK4, Cyclin D1, Bcl-2, but elevated the expression of p21, Bad and Bax. ICT1 was confirmed as a direct target of miR-1301-3p. Furthermore, ICT1 overexpression could partially reverse the effects of miR-1301-3p on BC cell proliferation, cell cycle progression and apoptosis. CONCLUSION: Our observations suggested that miR-1301-3p inhibits cell proliferation via inducing cell cycle arrest and apoptosis through targeting ICT1, and might be a therapeutic target for BC.

Hibernoma Mimicking Atypical Lipomatous Tumor: 64 Cases of a Morphologically Distinct Subset.

Hibernoma is a benign adipocytic tumor with predilection for subcutaneous tissue of the thigh, upper trunk, and neck of middle-aged adults. 11q13 rearrangement resulting in MEN1/AIP codeletion is characteristic. Hibernomas are composed, in varying proportions, of brown fat cells, mature adipocytes, and microvacuolated lipoblast-like cells. Examples containing predominantly multivacuolated lipoblast-like cells are uncommon and distinction from atypical lipomatous tumor (ALT) is important for clinical management. We herein present the clinicopathologic features of 64 hibernomas histologically mimicking ALT. MDM2 and CDK4 immunohistochemistry as well as MDM2 fluorescence in situ hybridization were performed in a subset of cases. Clinical and follow-up information were obtained from referring pathologists. Thirty-four patients were male and 30 female, with a median age of 43 years (range, 24 to 78 y). The tumors were well circumscribed and mostly deeply located (53/64 cases, 83%) with a median tumor size of 12.9 cm (range, 3.5 to 23 cm) and predilection for the thigh (42/64 cases, 66%). Histologically, large cells with prominent lipoblast-like cytoplasmic fatty vacuoles and small central nuclei were present to a prominent degree in all cases, along with mature univacuolated adipocytes and smaller numbers of large, finely vacuolated cells with eosinophilic granular cytoplasm. Nuclear atypia and mitoses were absent. None of the 39 cases tested showed CDK4 and MDM2 overexpression or MDM2 amplification. Follow-up, available for 16/64 cases (median, 47 mo; range, 1 to 165 mo), revealed no recurrences or metastases. Hibernoma mimicking ALT shows predilection for deep soft tissue, especially in the thigh. These tumors behave in a benign manner and MDM2/CDK4 negativity may be useful in excluding ALT.

LncRNA CCAT1 promotes the progression of preeclampsia by regulating CDK4.

OBJECTIVE: Preeclampsia is one of the leading causes of maternal and perinatal deaths. This study mainly explored the mechanism of long non-coding RNA (lncRNA) CCAT1 expression in the placenta of preeclampsia patients and its effect on the progression of preeclampsia. MATERIALS AND METHODS: We used quantitative reverse transcription PCR (qRT­PCR) to detect the lncRNA CCAT1 expression in 40 preeclampsia and 40 normal pregnancy placenta samples. CCAT1 expression and its relationship with the clinicopathological parameters of preeclampsia was statistically analyzed. The specific small interfering RNA (si-CCAT1) and plasmid (pcDNA-CCAT1) targeting lncRNA CCAT1 were synthesized and transfected into Bew and JEG-3 cells. The CCAT1 expression in Bew and JEG-3 cells was determined by qRT­PCR. The effect of overexpression and interference of lncRNA CCAT1 on the proliferation of Bew and JEG-3 cells was observed. The effect of CCAT1 on cell cycle was examined by cell cycle assay. The protein expression was accessed by Western blot. RESULTS: Higher lncRNA CCAT1 expression was found in preeclampsia patients. The systolic blood pressure, diastolic blood pressure and urine protein in preeclampsia patients were significantly higher than those in normal pregnant women. The birth weight of fetus was significantly lower than that of normal pregnant women. However, there was no significant difference in weight and age of patients. According to the CCAT1 expression, preeclampsia patients were assigned into high expression group and low expression group. Higher systolic blood pressure, diastolic blood pressure, and urinary protein levels in CCAT1 high expression group were observed comparing to those in low expression group, while the birth weight in low expression group was significantly higher than the high expression group. In addition, we found that after interference with CCAT1, trophoblast proliferation was significantly increased and cell cycle was significantly accelerated, whereas overexpression of CCAT1 led to the contrary. Western blotting indicated that the expressions of E2F1, cyclin D, CDK2 and CDK4 in BeWo cells were increased after CCAT1 was knocked down. The expressions of E2F1, cyclin D, CDK2 and CDK4 in JEG3 cells were decreased after CCAT1 was overexpressed. CONCLUSIONS: LncRNA CCAT1 was highly expressed in preeclampsia and can promote the progression of preeclampsia by inhibiting the expression of CDK4.

CDK4 expression in chordoma: A potential therapeutic target.

Chordomas are rare bone tumors and treatment is commonly based on a combination of surgery and radiotherapy. There is no standard chemotherapy treatment for chordoma. The aim of this study was to determine the expression of cyclin-dependent kinase 4 (CDK4) in chordoma and its therapeutic implications. We evaluated CDK4 expression both in chordoma cell lines and in chordoma tissues. Also, we investigated the functional roles of CDK4 in chordoma cell growth and proliferation. Furthermore, the therapeutic implications of targeting CDK4 in chordoma were evaluated. We found CDK4 highly expressed in chordoma cell lines and in a majority (97.7%) of chordoma tissues. Higher CDK4 expression correlated with metastasis and recurrence of chordoma. Treatment of chordoma cells using CDK4 inhibitor palbociclib could efficiently inhibit chordoma cells growth and proliferation. These data demonstrate that targeting CDK4 may be useful as a novel strategy in the treatment of chordoma. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1581-1589, 2018.

Lanthanide-based peptide biosensor to monitor CDK4/cyclin D kinase activity.

We describe a lanthanide biosensor that responds to CDK4 kinase activity in melanoma cell extracts through a significant and dose dependent increase in luminescence, thanks to sensitization of a DOTA[Tb3+] complex incorporated into a CDK4 substrate peptide by a unique tryptophan residue in an adjacent phosphoaminoacid binding moiety.

Primary dermal pleomorphic liposarcoma: utility of adipophilin and MDM2/CDK4 immunostainings.

Liposarcoma, usually arises in deep soft tissues and pleomorphic liposarcoma (PL), is the rarest histopathologic variant. However, 15 cases of entirely dermal PL have been reported. We describe a case of a 79-year-old man who developed a rapidly growing nodule on his thorax. Excisional biopsy was performed and immunohistochemical studies were carried. The lesion was a well-circumscribed dermal nodule composed of multivacuolated pleomorphic lipoblasts and atypical mitotic figures. Neoplastic cells expressed CD10 and resulted negative S100 protein, Melan-A, MITF-1, AE1/AE3, CD4, CD68 (PGM1), retinoblastoma gene family protein, pericentrine and lysozyme. Adipophilin stain showed the lipid contents in the cytoplasm of the neoplastic cells. MDM2 and CDK4 resulted both negative. A diagnosis of primary dermal PL was made. This case shows the utility of adipophilin immunostaining to prove the lipid contents in neoplastic cells, which has the advantage of using formalin-fixed paraffin-embedded tissue and making needless frozen sections and ultrastructural studies to show these findings. Negative MDM2/CDK4 staining in our case argues against the possibility of dedifferentiated liposarcoma and further supports the diagnosis of true PL.

Differential diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: utility of p16 in combination with MDM2 and CDK4 immunohistochemistry.

The differential diagnosis between atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS) from their morphologic counterparts is challenging. Currently, the diagnosis is guided by MDM2 and CDK4 immunohistochemistry (IHC) and is confirmed by the amplification of the corresponding genes. Recently, p16 IHC has been proposed as a useful diagnostic biomarker. The objective was to assess the utility of p16 IHC in the differential diagnosis of ALT/WDLPS and DDLPS. Our series included 101 tumors that were previously analyzed using fluorescence in situ hybridization for MDM2 and CDK4 amplification. We compared sensitivity and specificity of p16 IHC to MDM2 and CDK4 IHC in the differential diagnosis of ALT-WDLPS (n=19) versus benign adipocytic tumors (n=44) and DDLPS (n=18) versus mimicking sarcomas (n=20). In the differential diagnosis of ALT-WDLPS, p16 had a sensitivity of 89.5% but a specificity of 68.2%, which was impaired by false-positive lipomas with secondary changes, especially in biopsies. Likewise, in the differential diagnosis of DDLPS, p16 had a sensitivity of 94.4% and a specificity of 70%, which hampered its use as a single marker. However, adding p16 to MDM2 and/or CDK4 increased diagnostic specificity. Indeed, MDM2+/p16+ tumors were all ALT-WDLPS, and MDM2-/p16- tumors were all benign adipocytic tumors. Moreover, all MDM2+/CDK4+/p16+ tumors were DDLPS, and the MDM2-/CDK4-/p16- tumor was an undifferentiated sarcoma. Although the use of p16 as a single immunohistochemical marker is limited by its specificity, its combination with MDM2 and CDK4 IHC may help discriminate ALT-WDLPS/DDLPS.

Inhibition of heat shock protein 90 improves pulmonary arteriole remodeling in pulmonary arterial hypertension.

While the molecular chaperone heat shock protein 90 (HSP90) is involved in a multitude of physiological and pathological processes, its role relating to pulmonary arterial hypertension (PAH) remains unclear. In the present study, we investigated the effect in which HSP90 improves pulmonary arteriole remodeling, and explored the therapeutic utility of targeting HSP90 as therapeutic drug for PAH. By Elisa and immunohistochemistry, HSP90 was found to be increased in both plasma and membrane walls of pulmonary arterioles from PAH patients. Moreover, plasma HSP90 levels positively correlated with mean pulmonary arterial pressure and C-reactive protein. In a monocrotaline-induced rat model of PH, we found that 17-AAG, a HSP90-inhibitor, alleviated the progress of PH, demonstrated by lower pulmonary arterial pressure and absence of right ventricular hypertrophy. Immunohistochemical staining demonstrated that 17-AAG improved pulmonary arteriole remodeling on the basis of reduced wall thickness and wall area. The inflammatory response attributed to PH could be attenuated by 17-AAG through reduction of NF-κB signaling. Moreover, 17-AAG was found to suppress PDGF-stimulated proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through induction of cell cycle arrest in the G1 phase. In conclusion, HSP90 inhibitor 17-AAG could improve pulmonary arteriole remodeling via inhibiting the excessive proliferation of PASMCs, and inhibition of HSP90 may represent a therapeutic avenue for the treatment of PAH.

Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma.

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

UNLABELLED: The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. CONTROL: 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle.

Clinical outcome of low-grade central osteosarcoma and role of CDK4 and MDM2 immunohistochemistry as a diagnostic adjunct.

INTRODUCTION: Low-grade osteosarcoma encompasses parosteal osteosarcoma (POS) and low-grade central osteosarcoma (LCOS), with LCOS more rare than POS. LCOS is also more likely to be misdiagnosed and inappropriately treated with an intralesional procedure, due to its misleading radiological features and the overlap of its pathological characteristics with those of benign bone tumors. Therefore, as a diagnostic adjunct for LCOS, immunohistochemical assay with murine double-minute type 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) have been tried with controversial results. We investigated (1) the clinical course and surgical outcome of LCOS, and (2) the diagnostic role of immune-histochemical markers (CDK4, MDM2) and their correlation with clinico-radiologic findings. MATERIALS AND METHODS: We retrospectively reviewed 16 LCOS patients with regard to age, gender, tumor location, plain radiographic pattern, tumor volume, extraosseous extension, initial diagnosis, initial treatment, definitive diagnosis, definitive treatment, surgical margins, histochemical markers, and oncological outcome. RESULTS: Final survival status was continuous disease-free in 14, alive with disease in 1, and remaining 1 patient died of other cancer. Except for 1 patient who had not undergone excision of their primary lesion, no patients developed a local recurrence. Eight tumors (50%) showed diffuse immunostaining for CDK4. Three of 8 tumors labeled for CDK4 were also positive for MDM2. Six (75%) of 8 CDK4-positive tumors displayed lytic lesions on a plain radiograph; in contrast, 2 (33%) of 6 tumors showing a sclerotic pattern on a plain radiograph were positive for CDK4. CONCLUSIONS: The diagnosis of LCOS is challenging; however, if it is properly diagnosed, there is a high chance of a cure with wide excision alone. Positive immunostaining for CDK4 or MDM2 may be used as a diagnostic adjunct, although negative immunostaining cannot rule out this tumor. The clinical, radiological, and typical pathological findings are vital in raising the suspicion of this rare tumor.

Emodin azide methyl anthraquinone derivative induced G0/ G1 arrest in HER2/neu-overexpressing MDA-MB-453 breast cancer cells.

PURPOSE: Our previous data have shown that emodin azide methyl anthraquinone derivative (AMAD) triggered mitochondrial- dependent cell apoptosis involving caspase-8-mediated Bid cleavage, and induced proteasomal degradation of HER2/neu by blocking Her2/neu binding to Hsp90. In the present study, we futher investigated the effect of this compound on the cell cycle and related molecular mechanisms in HER2/neu-overexpressing MDA-MB-453 breast cancer cells. METHODS: The cell cycle distribution was tested by flow cytometry. The expression of cell cycle-related proteins was determined by Western blot analysis; DNA agarose gel electrophoresis was used to examine the apoptosis of MDAMB- 453 cells induced by emodin AMAD. RESULTS: After MDA-MB-453 cells were treated with different concentrations of emodin AMAD for 24 hrs, cells were arrested in G0/G1 phase, and the expression of G0/G1 related proteins c/Myc, Cyclin D1, CDK4 and p-Rb changed. DNA fragmentation appeared on the agarose gel in a concentration- dependent manner. CONCLUSION: Emodin AMAD induced G0/G1 arrest in Her2/ neu-overexpressing MDA-MB-453 cancer cells. This G0/G1 arrest was associated with decreasing protein expression of c-Myc, Cyclin D1, CDK4, and p-Rb.

Green tea extract inhibition of human leiomyoma cell proliferation is mediated via catechol-O-methyltransferase.

BACKGROUND/AIMS: To investigate the inhibitory effect of green tea extract, epigallocatechin gallate (EGCG), on wild-type human leiomyoma (WT-HuLM) cells and its potential action via catechol-o-methyltransferase (COMT) activity. METHODS: Cell proliferation of WT-HuLM and COMT gene-silenced HuLM (COMT-shRNA-HuLM) cells treated with 0 or 100 µM EGCG for 7 days was measured using the MTT method. Total RNA and protein were extracted from cells treated with 0 or 100 µM of EGCG for 48 h. Gene expression profiling was performed using Human Signal Transduction PathwayFinder. Proliferation cell nuclear antigen (PCNA), cyclin-dependent kinase 4 (Cdk4) and COMT protein levels were detected by Western blot analyses. COMT enzyme activity was evaluated by HPLC. RESULTS: EGCG-treated WT-HuLM cells showed significantly decreased COMT expression (p < 0.001) and enzyme activity (p < 0.05) compared to untreated WT-HuLM cells, while COMT-shRNA-HuLM cells showed no significant change. At 100 µM of EGCG, survival of WT-HuLM cells was significantly lower (p < 0.05) compared to COMT-shRNA-HuLM cells. EGCG treatment modulated multiple signaling pathways in WT-HuLM compared to untreated control, while changes were minimal or reversed in COMT-shRNA-HuLM cells. EGCG significantly decreased PCNA, Cdk4 and soluble COMT protein levels (p < 0.001) in WT-HuLM, but not in COMT-shRNA-HuLM cells. CONCLUSIONS: The antiproliferative and gene-modulating effects of EGCG on HuLM cells are mediated, at least partially, via its effect on COMT expression and enzyme activity.

Immunohistochemical analysis of expressions of RB1, CDK4, HSP90, cPLA2G4A, and CHMP2B is helpful in distinction between myxofibrosarcoma and myxoid liposarcoma.

The role and diagnostic efficacy of gene and protein products RB1, CDK4, CHMP2B, HSP90, and cPLA2G4A, all previously shown to be involved in tumor genesis and cell proliferation, were examined by immunohistochemical techniques in 32 cases of myxofibrosarcomas and 29 myxoid liposarcomas (all diagnosis had been confirmed by fluorescence in situ hybridization). HSP90 demonstrated strong nuclear and cytoplasmic positivity in all myxoid liposarcoma cases, while only 4 myxofibrosarcomas showed scattered HSP90 positivity. All but 4 cases of myxofibrosarcoma displayed strong positivity for cPLA2G4A, while only 2 myxoid liposarcoma cases were cPLA2G4A positive and both were CHMP2B negative. Overexpression of both cPLA2G4A and CHMP2B also suggested higher tumor grade. In conclusion, HSP90 and cPLA2G4A immunohistochemical stains are useful markers to distinguish myxofibrosarcoma from myxoid liposarcoma.