Human Calmodulin‐Like Protein (CLP) Expression in Oral Squamous Mucosa and in Malignant Transformation

Abstract

The purpose of this study was to test whether calmodulin-like protein (CLP) is expressed in normal human oral mucosal cells and if downregulation of CLP occurs in malignant transformation. Oral mucosal tissue was taken from three individuals in a double-blind manner. The samples were cut, measured, and homogenized. Total RNA was extracted and reverse transcribed. Each cDNA sample was subjected to polymerase chain reaction (PCR). PCR fragments were purified, cloned, and sequenced to verify the presence of CLP. Three oral mucosal tissue samples with biopsy-confirmed squamous cell carcinoma were obtained. These samples demonstrated regions of normal epithelial cells as well as invasive squamous cell carcinoma. One normal breast epithelial sample was also obtained for positive control. Sections were stained with an affinity-purified CLP antibody and counterstained with a diluted hematoxylin. Two observers evaluated the specimens for expression of CLP. Staining patterns and intensity were noted in normal oral mucosa, comparing them to the normal breast epithelium sample. Staining patterns and intensity were then observed in squamous tumor cells, comparing them to the patterns of benign squamous mucosa. CLP coding sequences were positively identified from the normal oral mucosal tissue samples by reverse transcription and polymerase chain reaction (RT-PCR) with 100% identity to the published CLP sequence (accession #M58026). In the three oral mucosa tissue samples with known squamous cell carcinoma, expression of CLP was readily detected in areas of normal oral mucosa, while a notable downregulation of CLP expression occurred in areas of malignant transformation. The staining intensity was equivalent to the staining seen in the benign breast epithelium used as a control. In the areas of squamous cell carcinoma, a decrease in CLP immunoreactivity occurred. There was a sharp contrast in staining quality and clarity between benign and malignant tissue. In the majority of the carcinoma regions, a complete lack of immunoreactivity was noted. The RNA for human CLP is found in normal oral mucosa. CLP expression is seen in normal oral mucosa with a downregulation of CLP expression in malignant transformation.