Similarity of folate receptor expression in UMSCC 38 cells to squamous cell carcinoma differentiation markers.

BACKGROUND: The folate receptor is a 38 to 39 kd glycoprotein attached to the cell membrane via a glycosylphosphatidylinositol anchor. It serves as the initiation point for receptor-coupled transport of folate in a process known as potocytosis. The receptor is overproduced by a number of malignant cell lines in vitro and in a large percentage of ovarian and uterine malignancies. Immunohistochemistry has shown the receptor to be expressed primarily in normal differentiated tissues such as choroid plexus, placenta, thyroid, and kidney. The receptor gene(s) has been mapped to the human chromosomal locus 11q13. PURPOSE: In order to better understand regulation of the synthesis of the receptor, we studied the expression and accumulation of the folate receptor in UMSCC 38 cells, a human malignant squamous cell carcinoma line with a karyotype that is amplified at the folate receptor gene locus. METHODS: Western blot analysis of octylglucoside-solubilized cell membranes was used to analyze expression of several proteins by UMSCC 38 cells grown in culture under varied conditions. Bands on autoradiographs, representing electrophoresed proteins detected by indirect antibody labeling and an enhanced chemiluminescence reaction, were quantitated by densitometry. RESULTS: The expression of the folate receptor was found to increase approximately fourfold as UMSCC 38 cells were grown to higher cell densities over increasing lengths of time, ranging from 3 to 9 days, in culture. The expression of K1 keratin protein, a known marker of differentiation in squamous cell carcinomas, was elevated to a similar degree (3.2-fold) between day 5 and day 9 in cultures of these cells. Expression of folate-receptor protein in UMSCC 38 cells was also found to be reduced approximately 10-fold when cells were exposed to 1 microM retinoic acid for 48 hours and increased approximately eightfold after a 5-day exposure to 3 microM hydrocortisone. CONCLUSIONS: We present evidence that synthesis of the folate receptor in UMSCC 38 cells is affected by the same pathway that changes the expression of some markers of differentiation in squamous cell carcinomas. IMPLICATIONS: The fact that folate receptor expression in these malignant cells can be manipulated using retinoids and steroids suggests that these hormones could modulate the efficiency of folate and antifolate uptake via the folate receptor and may enhance the usefulness of the receptor as a target for immunotherapy.

UMSCC38 cells amplified at 11q13 for the folate receptor synthesize a mutant nonfunctional folate receptor.

Some cells accumulate folate via a receptor-coupled process termed potocytosis. The folate receptor, a glycosyl phosphatidylinositol anchored M(r) 38,000-39,000 glycoprotein, is coded for by at least two genes (FR alpha and FR beta) at 11q13. The karyotype of UMSCC38, a human squamous cell carcinoma cell line, suggests that it may contain multiple copies of the folate receptor gene(s). Southern blot analysis confirms the presence of four to six copies. Using polymerase chain reaction methodology, Northern blot analysis, immunoblotting, and immunofluorescence, we find relatively limited expression of FR alpha and no FR beta in UMSCC38 cells when compared to nonamplified lines. Antigen is observed on the cell surface in a punctate pattern, and the protein is anchored via a glycosyl phosphatidylinositol anchor. Transport of 5-[methyl-3H]tetrahydrofolic acid is blocked by 5-methyltetrahydrofolic acid and probenecid, which suggests anion transport. Monensin, an inhibitor of potocytosis, and folic acid, a high-affinity ligand for the receptor, do not effectively block 5-methyltetrahydrofolic acid transport. Taken together, the results suggest that UMCSCC38 cells, although gene amplified, synthesize surprisingly small amounts of receptor and that receptor is nonfunctional. In order to establish further the nature of the receptor, 16 clones were obtained, and the complementary DNA was sequenced. Three mutations were found.

Identification of a point mutation in the folate receptor gene that confers a dominant negative phenotype.

UM-SCC-38 cells, a squamous cell carcinoma cell line of the head and neck, express limited amounts of folate receptor alpha antigen which is not capable of binding either folic acid or 5-methyltetrahydrofolic acid. Three distinct mutations in the open reading frame of the folate receptor were identified. We now show that the three mutants are nonfunctional with respect to folic acid binding because the protein products do not bind folate. Additionally, a study of MA104 cells (a receptor-positive cell line) transfected with each mutant was done. Expression of one mutant, FR-67, results in a dominant negative phenotype because folate binding is significantly reduced although membrane antigen is significantly increased. Coexpression of FR-67 and the normal protein in MA104 cells also results in large, bright clusters of receptor protein inside the cell around the nucleus when visualized using indirect immunofluorescence. These clusters are not found in cells that express either normal or FR-67 protein alone. In conclusion, this study provides the first evidence of a mutant folate receptor protein capable of affecting normal receptor function in a dominant negative manner.

Architectural changes in the proximal femur following prosthetic insertion: preliminary observations of an animal model.

A study was made of the changes occurring in the proximal femur following unilateral total hip replacement in fourteen sheep. Implants were observed 0-12 months post-operatively. Contralateral implants inserted at sacrifice served as controls. The paired specimens were cut into five transverse sections and stereologically analyzed. The following phenomena were seen during the post-operative year: (a) calcar resorption; (b) encapsulation of the prosthetic cement in fibrous sheath; (c) interruption of the trabecular network between the cement-stem system and the cortex; (d) increase in the cross-sectional moment of inertia of the cortical shell. These changes form a consistent pattern of biological responses to the implant, the details of which are important because they suggest failure mechanisms and demonstrate the inadequacy of simple analytical models based on static pictures of the bone-implant composite structure. Failure modes may depend on the initial degree of bone-cement interdigitation. Concentric layers of fibrous tissue surrounded by layers of dense bone were formed where interdigitation was not extensive. Where interdigitation was maximal, a band of bony resorption separated the cement-bone complex from the endosteal trabecular bone. Increased levels of bone-cement interdigitation would not appear to influence the ultimate outcome, but, rather, only the mode of failure of the prosthetic attachment.

Effect of prolonged walking on concrete on the knees of sheep.

Adult sheep were subjected to prolonged activity on hard surfaces by walking them daily on concrete and housing them on tarmac. Control sheep were walked on compliant wood chip surfaces and pastured. After two and a half years significant changes were seen in both the distal femoral articular cartilage and subchondral trabecular bone of the knee joints of the hard surface walkers. The hexosamine content of the articular cartilage in the hard surface walkers was lower and this decrease was more marked in the weight-bearing than in the non-weight-bearing areas of the knee. The trabecular pattern of the subchondral bone became significantly altered, with a marked change in trabecular structure acting to stiffen the tibio-femoral joint at th expense of the patello-femoral articulation. There was a substantial increase in the contiguity ratio of bone in the tibio-femoral area. The cortical thickness of the subchondral plate was increased in both the tibio-femoral and patello-femoral areas. We concluded that significant changes occur in both cartilage and bone as a result of prolonged walking on hard surfaces.