Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPVnegative cancers arising in the same tissue.

The atomic force microscope is broadly used to study the morphology of cells1–5 but it can also probe the mechanics of cells. It is now known that cancerous cells may have different mechanical properties than normal cells6–8 but the reasons for these differences are poorly understood9. Here we report quantitatively the differences between normal and cancerous human cervical epithelial cells by considering the brush layer on the cell surface. These brush layers, which consist mostly of microvilli, microridges, and cilia are important for interacting with the environment. Deformation force curves obtained from cells in vitro are processed according to the 'brush on soft cell model 10. We found that normal cells have brushes with one length while cancerous cells displayed long and short brushes with significantly different densities. The observed differences suggest that brush layers should be taken into account when characterizing the cell surface by mechanical means.

Infection with high-risk human papillomaviruses (HPV) is a major risk factor for development of cervical cancer. Expression of the HPV E6 and E7 oncoproteins increases in differentiating keratinocytesHuman papillomaviruses (HPVs) are small DNA tumor viruses that replicate in differentiating keratinocytes of the epidermis and anogenital tract (59). The E6 and E7 viral genes are expressed at low levels in proliferating basal cells, but transcription is activated as cells enter the terminal differentiation pathway (11, 16). E6 and E7 delay keratinocyte differentiation, reactivate host DNA synthesis, and stimulate cell cycle progression (60), allowing the virus to utilize host DNA synthetic enzymes to replicate its genome. HPVs induce benign warts and papillomas; however, infection with high-risk types ) is a major risk factor for the development of cervical cancer (reviewed in reference 60). The E6 and E7 genes are particularly important because they are retained and expressed in most cervical carcinomas, and continued expression is required to maintain the malignant phenotype (55).An early step in HPV-associated carcinogenesis is perturbation of cellular gene expression by the E6 and E7 oncoproteins.

Normal adult rat hepatocytes remained viable and functional for at least 43 days when plated on collagen-coated dishes and fed chemically defined medium supplemented with dimethyl sulfoxide (Me2SO). Hepatocytes isolated by collagenase perfusion and cultured in the presence or absence of Me2SO were (i) examined by light and electron microscopy for morphological changes; (it) analyzed for the production of albumin and other plasma proteins; and (iiM) tested by autoradiography for DNA synthesis. Me2SO-treated cells continued to produce specific plasma proteins during the entire culture period; albumin production was consistently high (11)(12)(13)(14)(15)(16)(17)(18)(19) ,.g/ml of culture medium per 24 hr) from day 2 to at least day 43 after plating. Ultrastructural analyses demonstrated that Me2SO-treated hepatocytes resembled those from intact liver in organization of cytoplasmic organelles and cellular junctions. The optimal concentration for observing the morphological and biochemical effects of Me2SO was 2% (vol/vol). We conclude that supplementation of chemically dermed medium with Me2SO enables maintenance ofdifferentiated hepatocytes in culture for extended periods of time.Dimethyl sulfoxide (Me2SO) is a dipolar aprotic solvent that is active in biological systems (1). Addition of 1-2% (vol/vol) Me2SO to the culture medium of Friend virus-induced murine erythroleukemia (MEL) cells for 4-5 days causes 90% of the cells to express characteristics associated with normal erythroid differentiation, including alterations in morphology (2), induction of a-and P-globin synthesis (3, 4), and loss of the capacity for cell division (5). Me2SO-induced differentiation has also been observed in a human leukemia cell line (6) and in cultured fibrosarcoma (7), neuroblastoma (8), human colon carcinoma (9), human lung cancer (10), and murine embryonal carcinoma (11, 12) cell lines.Past efforts to achieve long-term culture of differentiated normal adult hepatocytes have not been successful. Limited proliferation and maintenance of adult hepatocytes can be achieved by supplementing culture medium with serum from partially hepatectomized animals (13) or by plating hepatocytes on liver extracellular matrix and maintaining them in serum-free hormonally defined medium (14). Proliferation also can be achieved by culturing hepatocytes at low cell density in the presence of insulin and epidermal growth factor (EGF), but maintenance of hepatocyte-specific characteristics requires high density or supplementation with hepatic plasma membrane material (15,16).In the present study, we employed a collagen-coated surface and supplemented the culture medium with Me2SO in an attempt to extend the time in vitro that hepatocytes remain biochemically and morphologically differentiated. The addition of Me2SO had a dramatic effect; hepatocytes retaining morphological and biochemical characteristics of normal liver could be maintained in culture for as long as 43 days. Note that Me2SO, used previously to induce differentiation in tumor cells ...

The decrease in elasticity of epithelial tissues with ageing contributes to many human diseases. This change was previously attributed to increased crosslinking of extracellular matrix proteins. Here we show that individual human epithelial cells also become significantly more rigid during ageing in vitro. Using atomic force microscopy (AFM), we found that the Young's modulus of viable cells was consistently increased two-to four-fold in older versus younger cells. Direct visualization of the cytoskeleton using a novel method involving the AFM suggested that increased rigidity of ageing cells was due to a higher density of cytoskeletal fibres. Our results identify a unique mechanism that might contribute to the age-related loss of elasticity in epithelial tissues.