Targeted gene transfer system using a streptavidin-transforming growth factor-alpha chimeric protein.

The previously reported streptavidin-TGFalpha chimeric protein-based delivery system (Ohno and Meruelo, DNA Cell Biol. 15:401-406, 1996) could efficiently transfer protein molecules into A431 cells via the epidermal growth factor (EGF) receptor. We have modified this delivery system for the transfer of DNA. For this purpose, we have linked the chimeric protein ST-TGFalpha to DNA through biotinylated polylysine molecules. We show with this system, in the presence of the endosome-destabilizing reagent chloroquine, an average of 50-fold increase in reporter gene expression in comparison with polylysine DNA complexes alone. This gene expression is specific for EGF receptor-expressing cells and is blocked by EGF-binding molecules. These results suggest that the ST-TGFalpha biotinylated polylysine system could be used to deliver DNA to targeted cells.

P53 oncoprotein expression and gene mutations in some keratoacanthomas.

OBJECTIVE: To analyze the relationship of p53 oncoprotein overexpression in most keratoacanthomas (KAs) with gene mutations. DESIGN: Expression of p53 oncoprotein in immunohistochemical staining and its correlation to gene mutations in DNA extracted from KAs and tested in single-strand conformational polymorphism (SSCP) analysis and direct sequencing. SETTING: A micrographic surgery unit and a dermatopathology unit at a university medical center. PATIENTS: Sixteen formalin-fixed, paraffin-embedded skin biopsy specimens were retrieved from dermatopathology archives. Biopsies were performed to establish the diagnosis of KA before surgical treatment. MAIN OUTCOME MEASURES: Intensity of staining in immunohistochemical testing for p53 oncoprotein expression and sequencing of gene mutations. RESULTS: Immunohistochemical staining of 16 KA specimens detected p53 oncoprotein in 15 (94%), distributed as strong in 4 (25%), moderate in 2 (12%) mild in 9 (56%), and negative in 1 (6%), compared with control specimens. Specimens were screened by SSCP for mutations in the p53 gene, and 1 specimen showed a potential mutation in exon 7. Direct sequencing of the samples revealed 2 point mutations. One specimen showed a change of G:A for A:G in codon 146 of exon 5, predicting an amino acid substitution of tryptophan for a stop codon. Another specimen revealed a change of T:A for A:T in codon 234 of exon 7, predicting an amino acid substitution of tyrosine for asparagine. CONCLUSIONS: Ninety-four percent of KA specimens evaluated had detectable p53 oncoprotein. This protein was associated with a point mutation in the p53 gene in 2 of 16 KAs evaluated. In a small fraction of KAs, overexpression of p53 oncoprotein may be associated with point mutations in the p53 gene.

Primitive neuroectodermal tumors of the brainstem: investigation of seven cases.

OBJECTIVE: We discuss the clinical aspects, pathology, and molecular genetics of 7 patients with primitive neuroectodermal tumors (PNETs) arising in the brainstem that were treated at our institution from 1986 through 1995. Most neuro-oncologists avoid performing biopsies in children with pontine tumors. This article raises the question as to whether biopsies should be performed, because treatment recommendations might differ if a PNET was diagnosed rather than a pontine glioma. PATIENTS AND METHODS: We reviewed the clinical neuro-oncology database and the files of the Division of Neuropathology at New York University Medical Center from 1986 through 1995 and identified 7 histologically confirmed PNETs arising in the brainstem among 146 pediatric brainstem tumors. The clinical, neuroradiological, and neuropathological data were reviewed. Postmortem examinations were performed in 2 cases. Formalin-fixed, paraffin-embedded tumor tissues were also available in 6 of 7 patients that were tested for p53 gene mutations using single-strand conformation polymorphism analysis. We also tested 9 cerebellar PNETs, 9 brainstem gliomas, and 3 normal brains for p53 gene mutations as controls. RESULTS: All 7 patients presented with focal cranial nerve deficits, and 2 were also hemiparetic. The median age at diagnosis was 2.7 (1-8 years). Magnetic resonance imaging (MRI) characteristics included a focal intrinsic exophytic nonenhancing brainstem lesion that had low T1-weighted and high T2-weighted signals. Hydrocephalus was present in 5 patients at diagnosis, 3 of whom had leptomeningeal dissemination. Meningeal dissemination occurred later in the course of the disease in 3 other patients. Five children required shunts at diagnosis and another 2 at recurrence. Despite therapy, all 7 PNET patients died within 17 months of diagnosis with a mean survival of 8 (4-17) months. No mutation in the p53 gene was detected. CONCLUSIONS: Brainstem PNETs tend to arise at a younger age than brainstem gliomas and medulloblastomas. The MRI pattern suggests a localized rather than a diffuse intrinsic nonenhancing brainstem tumor. Like other PNETs, brainstem PNETs have a high predilection to disseminate within the central nervous system. The absence of p53 mutations is similar to other PNETs. Despite their origin close to the cerebellum, brainstem PNETs exhibit a more aggressive behavior and result in worse clinical outcomes than do cerebellar PNETs.