The role of p16 expression as a predictive marker in HPV-positive oral SCCHN--a retrospective single-center study.

UNLABELLED: Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common type of cancer worldwide; 600,000 new cases are diagnosed every year. Infected with high-risk human papilloma virus (HPV) types are particularly linked to oropharyngeal cancer. Among over 100 different HPV types, HPV-16 and HPV-18 are detected in the majority of HPV-positive SCCHNs. The p16 gene is often mutated in SCCHN, its overexpression is caused by the viral E7 protein. Consequently, p16 is assumed to be an indirect marker of HPV-induced SCCHN. The aim of the present study was to determine the role of p16 expression as a predictive marker of HPV infection in SCCHN tumors in a retrospective single-center study. MATERIALS AND METHODS: Oropharyngeal tumor samples from 45 patients (34 males, 11 females) were analyzed. Tumor samples were examined for HPV infection using a two-step PCR. p16 staining by immunohistochemistry was then performed. RESULTS: Samples with strong p16 signal were typed HPV-16-positive. Out of 14 tumor samples with HPV-positive PCR results, 13 samples contained the high risk variant HPV-16. In one sample, HPV-6 DNA was detected. All HPV-16-positive tumors overexpressed p16 (p16(+++)), whereas the HPV-6 sample was p16-negative. CONCLUSION: p16 is not a surrogate marker for replacing PCR testing, but both methods in combination, PCR and immunohistochemistry, could lead to a higher diagnostic validation.

Canalostomy as a surgical approach for cochlear gene therapy in the rat.

This article presents a unique approach for the delivery of gene therapy vectors into the cochlea of the laboratory rat. Mice and guinea pigs are established in vivo models for cochlear gene therapy each of which has distinct advantages and disadvantages. The rat has some of the molecular advantages of a mouse model combined with size advantages for surgical approaches. Vector delivery via cochleostomy or injection through the round window causes concomitant sensorineural hearing loss and is therefore not suitable for studies where the change in hearing is being followed. Compared to the mouse, the rat does not demonstrate easily recognizable landmarks that allow for use of the semicircular canal as an approach to the inner ear. We analyzed sagittal and coronal temporal bone sections of Long Evans rats and identified the bony entrance of the facial nerve as a crucial landmark for canalostomy. Auditory brainstem response and distortion product otoacustic emission measurements revealed minimal differences in the hearing threshold after adenovirus vector application when large volumes of vector were infused to the inner ear. Canalostomy and infusion of adenoviral vectors also resulted in temporary balance disturbance in the rat. Immunohistochemical assessment after delivery of a green fluorescent protein expressing vector showed significant GFP expression in the cochlea.

The challenge of tumor heterogeneity--different phenotypes of cancer stem cells in a head and neck squamous cell carcinoma xenograft mouse model.

BACKGROUND/AIM: Besides late diagnosis, tumor metastasis and cancer relapse are the main reasons for the poor prognosis of patients with head and neck cancer. Several investigations have shown that tumor is of heterogeneous molecularity consisting of several subpopulations, with a broad range of biological behaviors. The ability and potential of tumor to infiltrate into vessels and into neighbouring organs, as well as the resistance to chemotherapeutical cancer therapy may be caused by cancer stem cells (CSCs). The aim of the present study was to illuminate the role and behaviour of (CD44) and (ALDH1A1) as tumor stem cell markers in a xenograft mouse model of squamous cell carcinoma. MATERIALS AND METHODS: Five female NMRI-Foxn1nu mice were injected with five million Detroit 562 cells (100 µl). After sacrifice of the mice, tumors were excised. Then ALDH1A1, CD44, (EGFR), CD31 and Ki 67 were detected as molecular markers for tumor stem cells by immunohistopathology and immunofluorescence. RESULTS: The amount of putative CSC marker proteins CD44 and ALDH1A1 vary. ALDH1A1high tumor cells express low levels of CD44 and EGFR. The CD44+high expressers also exhibit expression of high levels of the EGFR. CSCs must be sub-classified depending on their expression of marker proteins. CONCLUSION: We assume that CSCs can also be sub-classified into migratory and stationary CSCs. ALDH1A1high/CD44low/EGFRlow tumor cells may be stationary and quiescent, whereas ALDH1A1-/CD44high/EGFRhigh expressers have a migratory, invasive nature. It is likely that a regulatory mechanism, as yet unknown, controls this conversion, from quiescent to active cancer stem cells.

Activation of classical protein kinase C reduces the expression of human cationic amino acid transporter 3 (hCAT-3) in the plasma membrane.

We have previously shown that activation of PKC (protein kinase C) results in internalization of hCAT-1 [human CAT-1 (cationic amino acid transporter 1)] and a decrease in arginine transport [Rotmann, Strand, Martiné and Closs (2004) J. Biol. Chem. 279, 54185-54192]. However, others found increased transport rates for arginine in response to PKC activation, suggesting a differential effect of PKC on different CAT isoforms. Therefore we investigated the effect of PKC on hCAT-3, an isoform expressed in thymus, brain, ovary, uterus and mammary gland. In Xenopus laevis oocytes and human U373MG glioblastoma cells, hCAT-3-mediated L-arginine transport was significantly reduced upon treatment with compounds that activate classical PKC. In contrast, inactive phorbol esters and an activator of novel PKC isoforms had no effect. PKC inhibitors (including the PKCalpha-preferring Ro 31-8280) reduced the inhibitory effect of the PKC-activating compounds. Microscopic analyses revealed a PMA-induced reduction in the cell-surface expression of fusion proteins between hCAT-3 and enhanced green fluorescent protein expressed in X. laevis oocytes and glioblastoma cells. Western-blot analysis of biotinylated surface proteins demonstrated a PMA-induced decrease in hCAT-3 in the plasma membrane, but not in total protein lysates. Pretreatment with a PKC inhibitor also reduced this PMA effect. It is concluded that similar to hCAT-1, hCAT-3 activity is decreased by PKC via reduction of transporter molecules in the plasma membrane. Classical PKC isoforms seem to be responsible for this effect.