Radiological progression of extremity soft tissue sarcoma following pre-operative radiotherapy predicts for poor survival.

OBJECTIVES: To determine if radiological response to pre-operative radiotherapy is related to oncologic outcome in patients with extremity soft tissue sarcomas (STSs). METHODS: 309 patients with extremity STS who underwent pre-operative radiation and wide resection were identified from a prospective database. Pre- and post-radiation MRI scans were retrospectively reviewed. Radiological response was defined by the modified Response Evaluation Criteria in Solid Tumours. Local recurrence-free, metastasis-free (MFS) and overall survival (OS) were compared across response groups. RESULTS: Tumour volume decreased in 106 patients (34.3%; PR - partial responders), remained stable in 97 (31.4%; SD - stable disease), increased in 106 (34.3%; PD - progressive disease). The PD group were older (p = 0.007), had more upper extremity (p = 0.03) and high-grade tumours (p < 0.001). 81% of myxoid liposarcomas showed substantial decrease in size. There was no difference in initial tumour diameter (p = 0.5), type of surgery (p = 0.5), margin status (p = 0.4), or complications (p = 0.8) between the three groups. There were 10 (3.2%) local recurrences with no differences between the three response groups (p = 0.06). 5-year MFS was 52.1% for the PD group vs 73.8 and 78.5% for the PR and SD groups, respectively (p < 0.001). OS was similar (p < 0.001). Following multivariable analysis, worse MFS and OS were associated with higher grade, larger tumour size at diagnosis and tumour growth following pre-operative radiation. Older age was also associated with worse OS. CONCLUSION: STS that enlarge according to Response Evaluation Criteria in Solid Tumour criteria following pre-operative radiotherapy identify a high risk group of patients with worse systemic outcomes but equivalent local control. ADVANCES IN KNOWLEDGE: Post-radiation therapy, STS enlargement may identify patients with potential for worse systemic outcomes but equivalent local control. Therefore, adjunct therapeutic approaches could be considered in these patients.

Paraneoplastic Dermatomyositis.

Dermatomyositis and its association with malignancy is well known. Though many tumors like breast, ovary, lung carcinomas are reported to be associated with it, cervical carcinoma is a very rare one. Tumors in these patients are usually unmasked by abnormal findings in the medical history and physical examination. Here we report case of a postmenopausal female who presented with subacute onset of weakness of all four limbs. Treatment of the underlying tumor relieved the symptoms of dermatomyositis in our patient.

Management of soft-tissue sarcomas; treatment strategies, staging, and outcomes.

Soft-tissue sarcomas (STS) are a rare group of malignant tumors which can affect any age group. For the majority of patients who present with a localized STS, treatment involves a multidisciplinary team decision-making approach ultimately relying on surgical resection with or without adjuvant radiation for successful limb salvage. The goals of treatment are to provide the patient with a functional extremity without local tumor relapse. The purpose of this article is to review the treatment of extremity STS, with a focus on staging, treatment options, and outcomes.

Cell Synchronization of Mouse Embryonic Fibroblasts.

A fundamental need in the analysis of the cell cycle is the ability to isolate relatively homogeneous populations of cells in different phases. This is complicated by the variable proliferative properties and responses to synchronizing methods of different cancer-derived cell lines. Paradoxically, cell lines with genetic defects in cell cycle control are sometimes chosen because they are amenable to chemical synchronization. Embryonic fibroblasts from mice present the opportunity to study the effects of defined genetic modifications on a normal cell cycle. However, synchronization of these cells has often been challenging. In this chapter we outline three basic protocols for isolating mouse fibroblasts at the G1-to-S-phase transition, in S phase, and during mitosis.

Notch Signaling is Associated with ALDH Activity and an Aggressive Metastatic Phenotype in Murine Osteosarcoma Cells.

Osteosarcoma (OS) is the most common primary malignancy of bone, and pulmonary metastatic disease accounts for nearly all mortality. However, little is known about the biochemical signaling alterations that drive the progression of metastatic disease. Two murine OS cell populations, K7M2 and K12, are clonally related but differ significantly in their metastatic phenotypes and therefore represent excellent tools for studying metastatic OS molecular biology. K7M2 cells are highly metastatic, whereas K12 cells display limited metastatic potential. Here we report that the expression of Notch genes (Notch1, 2, 4) are up-regulated, including downstream targets Hes1 and Stat3, in the highly metastatic K7M2 cells compared to the less metastatic K12 cells, indicating that the Notch signaling pathway is more active in K7M2 cells. We have previously described that K7M2 cells exhibit higher levels of aldehyde dehydrogenase (ALDH) activity. Here we report that K7M2 cell ALDH activity is reduced with Notch inhibition, suggesting that ALDH activity may be regulated in part by the Notch pathway. Notch signaling is also associated with increased resistance to oxidative stress, migration, invasion, and VEGF expression in vitro. However, Notch inhibition did not significantly alter K7M2 cell proliferation. In conclusion, we provide evidence that Notch signaling is associated with ALDH activity and increased metastatic behavior in OS cells. Both Notch and ALDH are putative molecular targets for the treatment and prevention of OS metastasis.

Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells.

Osteosarcoma (OS) is the most common primary malignancy of bone. Mortality is determined by the presence of metastatic disease, but little is known regarding the biochemical events that drive metastases. Two murine OS cell lines, K7M2 and K12, are related but differ significantly in their metastatic potentials: K7M2 is highly metastatic whereas K12 displays much less metastatic potential. Using this experimental system, the mammalian target of rapamycin (mTOR) pathway has been implicated in OS metastasis. We also discovered that aldehyde dehydrogenase (ALDH, a stem cell marker) activity is higher in K7M2 cells than K12 cells. Rapamycin treatment reduces the expression and enzymatic activity of ALDH in K7M2 cells. ALDH inhibition renders these cells more susceptible to apoptotic death when exposed to oxidative stress. Furthermore, rapamycin treatment reduces bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) gene expression and inhibits K7M2 proliferation, migration, and invasion in vitro. Inhibition of ALDH with disulfiram correlated with decreased mTOR expression and activity. In conclusion, we provide evidence for interaction between mTOR activity, ALDH activity, and metastatic potential in murine OS cells. Our work suggests that mTOR and ALDH are therapeutic targets for the treatment and prevention of OS metastasis.

Dystrophin and utrophin "double knockout" dystrophic mice exhibit a spectrum of degenerative musculoskeletal abnormalities.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. In addition, DMD patients acquire osteopenia, fragility fractures, and scoliosis indicating that a deficiency in skeletal homeostasis coexists but little is known about the effects of DMD on bone and other connective tissues within the musculoskeletal system. Recent evidence has emerged implicating adult stem cell dysfunction in DMD myopathogenesis. Given the common mesenchymal origin of muscle and bone, we sought to investigate bone and other musculoskeletal tissues in a DMD mouse model. Here, we report that dystrophin-utrophin double knockout (dko) mice exhibit a spectrum of degenerative changes, outside skeletal muscle, in bone, articular cartilage, and intervertebral discs, in addition to reduced lifespan, muscle degeneration, spinal deformity, and cardiomyopathy previously reported. We also report these mice to have a reduced capacity for bone healing and exhibit spontaneous heterotopic ossification in the hind limb muscles. Therefore, we propose the dko mouse as a model for premature musculoskeletal aging and posit that a similar phenomenon may occur in patients with DMD.

Intestinal infestation with Fasciolopsis buski leading to acute kidney injury.

Fasciolopsis buski is the largest intestinal fluke infecting human beings. This trematode is endemic in certain parts of the country. Migration poses the risk of spread of the worm to other parts of the country. We report fasciolopsis buski in a migrant from Bihar working in Coimbatore, Tamil Nadu. Acute kidney injury following intestinal obstruction occurred in this case which was never described before.

Biologic approaches to enhance rotator cuff healing after injury.

BACKGROUND: Despite the advances in surgical procedures to repair the rotator cuff, there is a high incidence of failure. Biologic approaches, such as growth factor delivery and stem cell and gene therapy, are potential targets for optimization to improve the outcome of rotator cuff therapies and reduce rates of reinjury. This article outlines the current evidence for growth factor and stem cell therapy in tendon healing and the augmentation of rotator cuff repair. METHODS: Literature on the PubMed-National Center for Biotechnology Information database was searched using the keywords growth factor, factor, gene therapy, stem cell, mesenchymal, or bone marrow in combination with rotator cuff, supraspinatus, or infraspinatus. Articles that studied growth factors or stem cells alone in rotator cuff repair were selected. Only 3 records showed use of stem cells in rotator cuff repair; thus, we expanded our search to include selected studies on stem cells and Achilles or patellar tendon repairs. Bibliographies and proceedings of meetings were searched to include additional applicable studies. We also included hitherto unpublished data by our group on the use of stem cell transplantation for rotator cuff therapy. RESULTS: More than 70 articles are summarized, with focus on recent original research papers and significant reviews that summarized earlier records. CONCLUSIONS: Use of growth factors, stem cell therapy, and other tissue-engineering means serve to augment classical surgical rotator cuff repair procedures. The combination of stem cells and growth factors resulted in enhanced repair that emulated uninjured tissue, but the literature search reflected paucity of research in this field. Preclinical evidence from gene therapy and stem cell studies can be used as a start to move therapy from the experimental phase to clinical translation in patients.

A G1 checkpoint mediated by the retinoblastoma protein that is dispensable in terminal differentiation but essential for senescence.

Terminally differentiated cell types are needed to live and function in a postmitotic state for a lifetime. Cellular senescence is another type of permanent arrest that blocks the proliferation of cells in response to genotoxic stress. Here we show that the retinoblastoma protein (pRB) uses a mechanism to block DNA replication in senescence that is distinct from its role in permanent cell cycle exit associated with terminal differentiation. Our work demonstrates that a subtle mutation in pRB that cripples its ability to interact with chromatin regulators impairs heterochromatinization and repression of E2F-responsive promoters during senescence. In contrast, terminally differentiated nerve and muscle cells bearing the same mutation fully exit the cell cycle and block E2F-responsive gene expression by a different mechanism. Remarkably, this reveals that pRB recruits chromatin regulators primarily to engage a stress-responsive G(1) arrest program.

A functional connection between pRB and transforming growth factor beta in growth inhibition and mammary gland development.

Transforming growth factor beta (TGF-beta) is a crucial mediator of breast development, and loss of TGF-beta-induced growth arrest is a hallmark of breast cancer. TGF-beta has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-beta cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1(DeltaL) and Rb1(NF)), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-beta growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-beta signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-beta cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-beta in growth control and mammary gland development.

Loss of ATRX leads to chromosome cohesion and congression defects.

Alpha thalassemia/mental retardation X linked (ATRX) is a switch/sucrose nonfermenting-type ATPase localized at pericentromeric heterochromatin in mouse and human cells. Human ATRX mutations give rise to mental retardation syndromes characterized by developmental delay, facial dysmorphisms, cognitive deficits, and microcephaly and the loss of ATRX in the mouse brain leads to reduced cortical size. We find that ATRX is required for normal mitotic progression in human cultured cells and in neuroprogenitors. Using live cell imaging, we show that the transition from prometaphase to metaphase is prolonged in ATRX-depleted cells and is accompanied by defective sister chromatid cohesion and congression at the metaphase plate. We also demonstrate that loss of ATRX in the embryonic mouse brain induces mitotic defects in neuroprogenitors in vivo with evidence of abnormal chromosome congression and segregation. These findings reveal that ATRX contributes to chromosome dynamics during mitosis and provide a possible cellular explanation for reduced cortical size and abnormal brain development associated with ATRX deficiency.

Enantiomeric resolution of supramolecular helicates with different surface topographies.

The enantiomeric resolution of an extended range of di-metallo supramolecular triple-helical molecules are reported. The ligands for all complexes are symmetric with two units containing an aryl group linked via an imine bond to a pyridine. Alkyl substituents have been attached in different positions on the ligand backbone. Previous work on the parent compound, whose molecular formula is [Fe(2)(C(25)H(20)N(4))(3)]Cl4, showed that it could be resolved into enantiomerically pure solutions using cellulose and 20 mM aqueous sodium chloride. In this work a range of mobile phases have been investigated to see if the separation and speed of elution could be increased and the amount of NaCl co-eluted with the compounds decreased. Methanol, ethanol and acetonitrile were considered, together with aqueous NaCl : organic mixtures. Effective separation was most often achieved when using 90% acetonitrile : 10% 20 mM NaCl (aq) w/v, which gives scope for scaling up to incorporate the use of HPLC. The overall most efficient (i.e. fastest) separation was generally achieved where the cellulose column was packed with 20 mM NaCl (aq) and the column first eluted with 100% acetonitrile, then with 75% ethanol : 25% 20 mM NaCl (aq) until the M enantiomer had fully eluted and finally with 90% acetonitrile : 10% 20 mM NaCl (aq) until the P enantiomer had been collected. The sequence of eluents ensured minimum NaCl accompanying the enantiomers and minimum total solvent being required to elute the enantiomers, especially the second one, from the column. No helicate with a methyl group on the imine bond could be resolved and methyl groups on the pyridine rings also have an adverse effect on resolution.

The retinoblastoma protein regulates pericentric heterochromatin.

The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G(1)-to-S-phase transition.

Intramolecular DNA coiling mediated by metallo-supramolecular cylinders: differential binding of P and M helical enantiomers.

We have designed a synthetic tetracationic metallo-supramolecular cylinder that targets the major groove of DNA with a binding constant in excess of 10(7) M(-1) and induces DNA bending and intramolecular coiling. The two enantiomers of the helical molecule bind differently to DNA and have different structural effects. We report the characterization of the interactions by a range of biophysical techniques. The M helical cylinder binds to the major groove and induces dramatic intramolecular coiling. The DNA bending is less dramatic for the P enantiomer.

Intramolecular DNA Coiling Mediated by a Metallo-Supramolecular Cylinder.

In the groove! A tetracationic supramolecular cylinder, [Fe2 L3 ]4+ (L=C25 H20 N4 ), with a triple-helical architecture is just the right size to fit into the major groove of DNA and too big to fit into the minor groove. NMR spectroscopic data confirm that the cylinder binds in the major groove. Linear dichroism shows that very low loadings of [Fe2 L3 ]4+ have a dramatic bending effect on the DNA and atomic force microscopy images show that this is an intramolecular effect resulting in coils of DNA.