Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: Implications for tumor biology

Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2,000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (i) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (ii) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.

p53 associates with and targets ΔNp63 into a protein degradation pathway

A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27–29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or ΔN-, respectively). The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of ΔNp63 (p40) led to increased growth of transformed cells in vitro and in vivo. Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia. We now demonstrate that certain p63 isotypes form complexes with p53. p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect. Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association. Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain ΔNp63 proteins (p40 and ΔNp63α). The association between p53 and ΔNp63 supports a previously unrecognized role for p53 in regulation of ΔNp63 stability. The ability of p53 to mediate ΔNp63 degradation may balance the capacity of ΔNp63 to accelerate tumorigenesis or to induce epithelial proliferation.

Skp2 is oncogenic and overexpressed in human cancers

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin–protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G1 progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-RasG12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Cloning and sequencing of CATR1.3, a human gene associated with tumorigenic conversion.

The human squamous cell carcinoma cell line SCC83-01-82 (SCC) contains mutations in both the H-ras and p53 genes, but it exhibits a nontumorigenic phenotype in nude mice. This cell line can be converted into a cell line with a tumorigenic phenotype, SCC83-01-82CA (CA), by treatment with the mutagen methyl methanesulfonate (MMS). This indicates that additional genetic events leading to expression of a cooperating tumor susceptibility gene(s) may be required for tumorigenicity. To identify the cooperating gene(s), an expression cDNA library was made from tumorigenic Ca cells. The library DNA was transfected into nontumorigenic SCC cells and the transfected SCC cells were then injected into nude mice for the selection of a tumorigenic phenotype. Tumors developed in 3 of the 18 mice after injection. Several new cell lines were established from these transfected cell-induced tumors and designated as CATR cells. Tumor histology and karyotype analysis of these cells indicated that they were of human epithelial cell origin. All the CATR cells have the library vector sequence integrated in their genome. Cell line CATR1 expressed a single message from the integrated library representing a 1.3-kb cDNA insert that was absent from untransfected SCC cells or MMS-converted CA cells. This 1.3-kb cDNA insert was cloned by PCR amplification of reverse-transcribed CATR1 total RNA and was designated CATR1.3. The nucleotide sequence of CATR1.3 encodes a peptide of 79 amino acids, has a long 3' untranslated region, and represents an unknown gene product that was associated with the tumorigenic conversion due to the transfected expression library.