Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization.

Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and is closely associated with infection of Epstein-Barr virus (EBV). The EBV encoded latent membrane protein 1 (LMP1) is frequently detected in NPC and may play a role in its pathogenesis. Previous studies have shown that LMP1 transformed rodent fibroblasts and altered growth properties in B cells and epithelial cells. However, the pathological role of LMP1 in NPC cells is still poorly understood. In order to investigate the downstream target genes of LMP1 in NPC cells, suppression subtractive hybridization was used to clone and identify the genes differentially expressed in a LMP1 expressing NPC cell line, CNE-2. Two subtractive cDNA libraries were constructed: one enriched for the genes upregulated by LMP1 and one was for the genes downregulated by LMP1. A total of 192 clones were screened by reverse Northern blotting. Fourteen of them were confirmed to be overexpressed while eight of them were suppressed. The upregulation of integrin alpha6, laminin 5gamma2, TAP1 and downregulation of p54nrb, RACK1 and p66Shc were further confirmed in three sets of LMP1 expressing NPC cell lines. The expression profiles of differentially expressed genes identified in this study suggest a role of LMP1 in promotion of cell survival and facilitation of tumor invasion.

Downregulation of hemidesmosomal proteins in nasopharyngeal carcinoma cells.

Hemidesmosome (HD) is a transmembrane complex that mediates attachment of epithelial cells to the basement membrane. Abnormal expression of HD components has been reported in several types of human cancers and is believed to play a role in tumor invasion and metastasis. Using differential gene display, we have identified downregulation of BPAG1 expression in nasopharyngeal carcinoma cells. BPAG1 is a major component of hemidesmosome. In the present study, we have extended our work to investigate the expression pattern of other components in the HD complex, namely, BPAG2, ITGalpha6 and ITGbeta4 in three distinct biological groups of nasopharyngeal epithelial cells: (a) non-malignant nasopharyngeal epithelial cells established from primary culture of nasopharyngeal explants, (b) non-malignant nasopharyngeal epithelial cells immortalized by viral oncogenes, SV40 or HPV16E6E7, and (c) nasopharyngeal carcinoma (NPC) cells. Both non-malignant primary cultured nasopharyngeal epithelial cells and immortalized nasopharyngeal epithelial cell lines expressed all the HD components examined, although the immortalized cells expressed a lower level of HD components compared with the non-malignant nasopharyngeal cells established from primary culture. In contrast, downregulation of HD components is commonly observed in nasopharyngeal carcinoma cells. Loss of HD expression in NPC may be associated with the undifferentiated properties of NPC cells and may have prognostic significance.

Differential gene expression in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a common cancer among southern Chinese. The profile of gene expression in NPC cells is largely unknown. In this study, we have examined differential gene expression in non-malignant and malignant nasopharyngeal epithelial (NPE) cells using a cDNA array hybridization method. A total of 42 genes were identified to be expressed in either non-malignant and malignant NPE cells or both. Thirteen of these genes were overexpressed in malignant NPE cells. These includes nuclear factor (NF90), FOS-related antigen 1 (FRA- 1), cytoplasmic dynein light chain (HDLC1), replication factor C (RFC1), nucleoside diphosphate kinase B, UV excision repair protein (RAD23A), insulin-like growth factor receptor II, transcription initiation factor TFIID subunit (TAFII31), growth factor receptor-bound protein 2 (GRB2), UV excision repair protein (RAD23B), glutathione peroxidase, Y box binding protein 1 and heat shock protein 86. In contrast, expression of nine genes was suppressed in malignant NPE cells. These includes calgranulin A, calgranulin B, neutrophil activating protein (ENA-78), heat shock protein 27, integrin beta-1, integrin beta-4, cyclin-dependent kinase inhibitor 1A (p21), interleukin-8 and tyrosine protein kinase receptor (RET). Differential expression of calgranulin A, calgraunlin B, ENA-78, FRA-1 and NF90 in non-malignant and malignant nasopharyngeal epithelial cells was confirmed by RT-PCR analysis.

Mechanism of differential sensitivity to cisplatin in nasopharyngeal carcinoma cells.

Cisplatin is used in the treatment of many tumours, including nasopharyngeal carcinoma (NPC). In this study, we studied two nasopharyngeal cancer cell lines with a four-fold difference in sensitivity to cisplatin. Following exposure to cisplatin, the sensitive SUNE1 cell line underwent apoptosis while the relatively resistant CNE1 line died through mitotic cell death. No differences were seen in telomere length or in the cell cycle distribution after cisplatin treatment. However, there was an increase in Bax levels in the sensitive cell line SUNE1, while in the resistant line CNE1 that did not undergo apoptosis, Bax levels fell. Our results suggest that upregulation of Bax is associated with the sensitivity of these NPC cells to cisplatin.

Pericranial healing and the temporalis myo-osseous flap in the rabbit model.

The purpose of this animal study was to determine the rate of revascularization of a temporalis myo-osseous (TMO) flap after pericranial elevation. In 24 rabbits, the right pericranium was raised in entirety through a bicoronal flap at the first operation. The pericranium was then reapproximated in situ. The pericranium was allowed to heal for 1 to 28 days before the second operation. At the second operation, through the same bicoronal flap, right and left temporalis myo-osseous flaps were raised. The left temporalis myo-osseous flap served as a control. Revascularization and viability of the temporalis myo-osseous flaps were studied by using technetium bone scans, india ink injection studies, and histologic study. Results demonstrated that 4 days following pericranial elevation, the temporalis myo-osseous flap is viable and revascularized by the pericranium. Immediate bone scanning and india ink injection showed patent pericranial circulation to the osseous portion of the temporalis myo-osseous flap at 4 days. Histologic study confirmed the viability of the temporalis myo-osseous flap. In conclusion, after pericranial elevation, pericranial healing and revascularization were complete at 4 days. This allowed a viable temporalis myo-osseous flap to be raised successfully at this time.

The role of tissue expanders in an anophthalmic animal model.

A study of orbital bony expansion using a custom tissue expander was performed in the anophthalmic cat model. Twelve 6-week-old kittens underwent right unilateral enucleations. Six kittens had immediate insertion of a tissue expander into the orbit. The remaining six served as controls. Every 2 weeks 0.5 cc saline was injected into the expander to a maximum of 5 cc. External horizontal and vertical orbital dimensions were obtained by palpation technique weekly. All animals had preoperative and study conclusion head CT scans with three-dimensional reconstructions performed. Dry skull preparations were done at the study conclusion at 24 weeks. Results demonstrated that tissue expanders were successful in maintaining normal orbital growth and size relative to the contralateral control orbit. The animals with enucleation only had an average difference in vertical and horizontal orbital measurements of -27 and -13 percent when compared with the contralateral normal orbit. In contrast, the enucleation and tissue-expansion animals had vertical and horizontal measurements of +4 and +2 percent (p less than 0.05) when compared with the contralateral orbit. Head CT scans with three-dimensional reconstructions demonstrated normal orbital geometry and volume for the animals with tissue expanders, whereas animals with enucleation only had small hypoplastic orbits. In conclusion, orbital tissue expanders offer a promising new technique in the treatment of anophthalmos.

Arch bar stabilization of endotracheal tubes in children with facial burns.

Endotracheal tube stabilization in children with facial burns can be difficult. Several methods rely on straps or complex devices that apply undesirable pressure to the face, potentially destroying skin grafts and making wound care difficult. Techniques that rely on a single wire or suture can be unreliable. Presented here is the arch bar method of endotracheal tube stabilization, which appears to be free of these problems. This method employs a standard dental arch bar secured to four maxillary teeth with 24-gauge stainless steel wire. The endotracheal tube is anchored to the arch bar with two pieces of wire or suture material. The arch bar method of endotracheal tube stabilization was used on three patients in the burn center at St. Christopher's Hospital for Children. Wound care and successful skin grafting were performed without difficulty. No complications related to the arch bars occurred.

Surgical correction of sagittal craniosynostosis: complications of the pi procedure.

We have completed a retrospective analysis of our experience with the pi procedure to determine the complications of the technique. Forty-five children underwent correction of scaphocephaly during the last 4 years. The average patient age was 9.2 months. The modified prone position with beanbag head support was used in 40 of 45 patients. Accompanied by barrel-stave and radial osteotomies, the reverse procedure was performed in 18 patients and frontal pulling in 27 patients. The average anteroposterior skull reduction was 1.44 cm, and mean blood loss was 96 ml, 11 ml/kg, replaced in 36 patients. Complications included three dural injuries, and air embolism was not detected by Doppler or end-tidal monitoring. A single child had a postoperative seizure. We conclude that complications of the pi procedure are limited and compare favorably with other repair techniques. We consider the pi procedure the technique of choice for correction of sagittal craniosynostosis in patients older than 3 months.

Cushing's syndrome in postburn children following intralesional triamcinolone injection.

Intralesional triamcinolone acetonide has been used extensively for the treatment of hypertrophic and keloid scars. Complications are few, usually being local skin color changes, prominent vascular markings, or subcutaneous atrophy. Cushing's syndrome following intralesional administration of triamcinolone acetate has only been described twice, both in adult patients. This paper reports on two pediatric cases of Cushing's syndrome following treatment of hypertrophic burn scars with intralesional triamcinolone acetonide. The first child, 10 years of age, was treated for hypertrophic burn scars of the back. The second child, age 21 months, was treated for hypertrophic burn scars of the face. In both, intralesional triamcinolone acetate therapy was initiated 3 months postburn injury and in neither child was the maximum recommended dose exceeded. In both children, Cushing's manifestations developed 1 to 2 weeks postinjection and resolved in 6 to 8 weeks with no treatment or permanent sequelae. Consultation with endocrinologists revealed no concurrent disease process. It appears that these two pediatric patients may have had a form of hypersensitivity to triamcinolone acetonide, as Cushing's syndrome was not the result of an overdose. In conclusion, intralesional triamcinolone acetonide should be used with an increased degree of caution in the pediatric population.

Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) occurs with a high incidence in many countries in south-eastern Asia. Chromosomal abnormalities have been commonly found in NPC, but the underlying mechanism is not well understood. We determined mitotic indices, the staining pattern of nuclear DNA and cell cycle profiles of NPC cells in response to treatment with microtubule-disrupting agents, and found that the mitotic checkpoint was defective in two out of five (40%) of the tested NPC cell lines. We also observed that an aberrantly reduced expression of MAD2, one of the key components of mitotic checkpoint, correlated with the loss of checkpoint control. Our findings suggest that a defective mitotic checkpoint characterized by reduced expression of MAD2 contributes to chromosomal instability in NPC.