Emerging of fractal geometry on surface of human cervical epithelial cells during progression towards cancer.

Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation.

Ultrabright fluorescent mesoporous silica nanoparticles for prescreening of cervical cancer.

We report on the first functional use of recently introduced ultrabright fluorescent mesoporous silica nanoparticles, which are functionalized with folic acid, to distinguish cancerous and precancerous cervical epithelial cells from normal cells. The high brightness of the particles is advantageous for fast and reliable identification of both precancerous and cancerous cells. Normal and cancer cells were isolated from three healthy women and three cancer patients. Three precancerous cell lines were derived by immortalization of primary cultures of normal cells with human papillomavirus type-16 (HPV-16) DNA. We observed substantially different particle internalization by normal and cancerous/precancerous cells after a short incubation time of 15 minutes. Compared to HPV-DNA and cell pathology tests, which are currently used for prescreening of cervical cancer, we demonstrated that the specificity of our method was similar (94-95%), whereas its sensitivity was significantly better (95-97%) than the sensitivity of those currently used tests (30-80%). FROM THE CLINICAL EDITOR: This team of investigators reports on the development of a new screening test for cervical cancer using ultrabright fluorescent mesoporous silica nanoparticles functionalized with folic acid, enabling significantly better sensitivity (95-97% vs. 30-80%) and maintained specificity (94-95%) compared with current clinical tests. This test should find a way to clinical use in the near future.

Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.

Cell surface as a fractal: normal and cancerous cervical cells demonstrate different fractal behavior of surface adhesion maps at the nanoscale.

Here we show that the surface of human cervical epithelial cells demonstrates substantially different fractal behavior when the cell becomes cancerous. Analyzing the adhesion maps of individual cervical cells, which were obtained using the atomic force microscopy operating in the HarmoniX mode, we found that cancerous cells demonstrate simple fractal behavior, whereas normal cells can only be approximated at best as multifractal. Tested on ~300 cells collected from 12 humans, the fractal dimensionality of cancerous cells is found to be unambiguously higher than that for normal cells.

Atomic force microscopy detects differences in the surface brush of normal and cancerous cells.

The atomic force microscope is broadly used to study the morphology of cells, but it can also probe the mechanics of cells. It is now known that cancerous cells may have different mechanical properties to those of normal cells, but the reasons for these differences are poorly understood. 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 mainly of microvilli, microridges and cilia, are important for interactions with the environment. Deformation force curves obtained from cells in vitro were processed according to the 'brush on soft cell model'. We found that normal cells have brushes of one length, whereas cancerous cells have mostly two brush lengths of significantly different densities. The observed differences suggest that brush layers should be taken into account when characterizing the cell surface by mechanical means.

Visualization of cytoskeletal elements by the atomic force microscope.

We describe a novel application of atomic force microscopy (AFM) to directly visualize cytoskeletal fibers in human foreskin epithelial cells. The nonionic detergent Triton X-100 in a low concentration was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized in either liquid or air-dried ambient conditions. These two types of scanning provide complimentary information. Scanning in liquid visualizes the surface filaments of the cytoskeleton, whereas scanning in air shows both the surface filaments and the total "volume" of the cytoskeletal fibers. The smallest fibers observed were ca. 50 nm in diameter. The lateral resolution of this technique was ca.20 nm, which can be increased to a single nanometer level by choosing sharper AFM tips. Because the AFM is a true 3D technique, we are able to quantify the observed cytoskeleton by its density and volume. The types of fibers can be identified by their size, similar to electron microscopy.

Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes.

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 keratinocytes, resulting in inactivation of the p53 and retinoblastoma proteins, two important transcriptional regulators. We used cDNA microarrays to examine global alterations in gene expression in differentiating cervical keratinocytes after infection with retroviruses encoding HPV type 16 (HPV-16) E6 and E7. Expression of 80 cellular genes (approximately 4% of the genes on the array) was altered reproducibly by E6 and/or E7. Cluster analysis classified these genes into three functional groups: (i) interferon (IFN)-responsive genes, (ii) genes stimulated by NF-kappaB, and (iii) genes regulated in cell cycle progression and DNA synthesis. HPV-16 E6 or a dominant negative p53 protein downregulated multiple IFN-responsive genes. E6 decreased expression of IFN-alpha and -beta, downregulated nuclear STAT-1 protein, and decreased binding of STAT-1 to the IFN-stimulated response element. E7 alone was less effective; however, coexpression of E6 and E7 downregulated IFN-responsive genes more efficiently than E6. The HPV-16 E6 protein also stimulated expression of multiple genes known to be inducible by NF-kappaB and AP-1. E6 enhanced expression of functional components of the NF-kappaB signal pathway, including p50, NIK, and TRAF-interacting protein, and increased binding to NF-kappaB and AP-1 DNA consensus binding sites. Secretion of interleukin-8, RANTES, macrophage inflammatory protein 1alpha, and 10-kappaDa IFN-gamma-inducible protein were increased in differentiating keratinocytes by E6. Thus, high-level expression of the HPV-16 E6 protein in differentiating keratinocytes directly alters expression of genes that influence host resistance to infection and immune function.

Microarrays: spotlight on gene function and pharmacogenomics.

The introduction of microarray technology has dramatically changed the way that researchers address many biomedical questions. DNA microarrays can measure expression of thousands of genes simultaneously, providing extensive information on gene interaction and function. Microarray technology is a powerful tool for identifying novel molecular drug targets and for elucidating mechanisms of drug action. Furthermore, microarrays can monitor the global profile of gene expression in response to specific pharmacologic agents, providing information on drug efficacy and toxicity. Over the last several years, dramatic advancements have occurred in array technology. In this review we describe basic aspects of microarray instrumentation and experimentation. Each of the major array formats including oligonucleotides arrays, spotted arrays, and macroarrays are examined, and advantages and options for using each format are presented. Important factors in the design and analysis of microarray experiments are also discussed. Most importantly, we explore recent developments in microarray technology that are relevant to pharmacogenomics and the discovery of gene function.

Targeted disruption of the epidermal growth factor receptor inhibits development of papillomas and carcinomas from human papillomavirus-immortalized keratinocytes.

The epidermal growth factor receptor (EGF-R) is frequently overexpressed in human papillomavirus (HPV)-associated dysplasias and carcinomas, implying that it is important for the progression of keratinocytes to malignancy. We used mice with a targeted disruption of the EGF-R gene to directly examine its role in cell immortalization and tumor development. Epidermal keratinocytes were cultured from EGF-R knockout, heterozygous, and wild-type mice, infected with retroviruses encoding HPV-16 E6 and E7 oncogenes, and grafted to nude mice. E6/E7 induced immortalization of EGF-R wild-type cells 5-fold more efficiently than null cells. Immortal EGF-R null cells grew more slowly, achieved a lower saturation density, and were more sensitive to apoptosis than the immortalized wild-type or heterozygous cells. Analyses using cDNA expression arrays showed that EGF-R null cells expressed increased levels of RNAs encoding p21waf and insulin-like growth factor-binding protein-2. EGF-R-positive immortal keratinocytes formed papillomas in 17% (15 of 90) of skin grafts, and seven grafts progressed to squamous carcinoma after 6-12 months. EGF-R null keratinocytes did not form papillomas, but 1 of 96 grafts progressed to a squamous carcinoma after 1 year. However, treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate induced tumors in 18 and 35% of grafts containing EGF-R null or EGF-R-positive cells, respectively. Transduction with an activated v-Ha-ras gene, which signals downstream of the EGF-R, induced rapidly growing carcinomas in all grafts regardless of EGF-R genotype. These results directly show that the EGF-R is important, but not essential, for immortalization by HPV and for progression of immortal cells to papillomas and carcinomas.

Human papillomavirus type 16 E6 and E7 proteins inhibit differentiation-dependent expression of transforming growth factor-beta2 in cervical keratinocytes.

Infection with high-risk human papillomaviruses (HPVs) represents a major risk factor for the development of cervical cancer. The HPV-16 E6 and E7 proteins are highly expressed in differentiating keratinocytes, where they inactivate the p53 and retinoblastoma (pRb) proteins, two important transcriptional regulators. We have used cDNA expression arrays to identify global alterations in gene expression induced by E6 and E7 in differentiating cultures of human cervical keratinocytes. We show that E6 and E7 decrease expression of TGF-beta2 mRNA and alter expression of multiple TGF-beta-responsive genes involved in cell cycle regulation, apoptosis, and tissue remodeling. E6 and E7 inhibited expression of TGF-beta2 RNA 7-fold (relative effectiveness, E6/ E7 > E6 > E7 > control) and decreased secretion of biologically active TGF-beta2 by 70-80% (reduced from 70 to 10 pg/10(6) cells/24 h). Downregulation occurred through p53- and pRb-dependent pathways. In contrast, E6 and E7 did not alter expression of TGF-beta1 and TGF-beta3. Down-regulation of TGF-beta2 was biologically relevant because the addition of recombinant cytokine (10-200 pg/ml) to E6/E7-expressing cells restored expression of TGF-P-responsive genes, inhibited growth of keratinocytes, and decreased immortalization by E6 and E7. These results suggest that TGF-32- and TGF-3-responsive genes are important targets for the HPV-16 E6 and E7 oncoproteins in differentiating cervical keratinocytes.

Tumor necrosis factor-alpha promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by a ras-dependent pathway.

Tumor necrosis factor-alpha (TNF-alpha) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)-immortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifies the mechanism of growth stimulation. TNF-alpha promoted cell cycle progression by increasing expression of HPV-16 E6/E7 RNAs and enhancing activity of cyclin-dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p21(waf) and p27(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activated Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited growth. Thus, activation of ras promotes expression of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates growth stimulation of immortal keratinocytes by TNF-alpha. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. 27:97-109, 2000. Published by Wiley-Liss, Inc.

Inhibitory effect of Bcl-2 on p53-mediated transactivation following genotoxic stress.

In the cellular response to genotoxic stress, cell cycle checkpoint and apoptosis are considered to be two of the major biological events in maintaining genomic stability. The tumor suppressor p53 has been shown to play critical roles in these stress-induced cellular responses at least in part through the activation of its down-stream genes, such as p21CIP1/WAF1, GADD45 and BAX. In addition, p53 has been found to down-regulate the expression of BCL-2, which is able to block apoptosis induced by both p53-dependent and independent signaling events. In this report, we have found that increased expression of Bcl-2 protein in the human Burkitt's lymphoma WMN cell line suppressed apoptosis induced by different DNA-damaging agents. The induction of p53-regulated genes including GADD45, p21CIP1/WAF1 and BAX by genotoxic stress was substantially reduced in cells expressing high levels of Bcl-2 protein. Furthermore, Bcl-2 protein was shown to specifically suppress the p53-mediated transactivation of p21CIP1/WAF1 and PG13-CAT, which is a typical p53-binding-site reporter construct. Similarly, the inhibitory effect of Bcl-2 protein was seen in a GADD45 promoter reporter construct after treatment with methylmethane sulfonate or UV-radiation. These results indicate that in addition to its apoptosis-suppressing activity, Bcl-2 protein is able to inhibit transactivation of p53-regulated genes, which function in multiple important cellular responses to genotoxic stress, including the control of cell cycle checkpoints, cell growth suppression and DNA repair.

Combination therapy with podophyllin and vidarabine for human papillomavirus positive cervical intraepithelial neoplasia.

Our aim was to establish an effective non-surgical treatment for cervical intraepithelial neoplasia (CIN) through inactivation of human papillomavirus (HPV), the major etiological agent for this disease. We show that vidarabine, a DNA polymerase inhibitor, suppressed growth and HPV gene expression in human cervical keratinocytes immortalized by HPV or in cervical cancer cell lines. Expression of HPV-16 E6 and E7 proteins in normal cervical keratinocytes sensitized cells to apoptosis in the presence of podophyllin or vidarabine. We applied vidarabine ointment and/or podophyllin to cervical epithelium in 28 cases of CIN I-II to evaluate the therapeutic effectiveness of these agents. Co-application of vidarabine and podophyllin in six treatments caused regression of lesions cytologically and histologically, and disappearance of HPV-16 or -18 DNA in 17 of 21 (81%) women. Our results suggest that the combination of vidarabine and podophyllin therapy is an effective non-surgical treatment for HPV-positive CIN.

Human papillomavirus type 16 E7 protein sensitizes cervical keratinocytes to apoptosis and release of interleukin-1alpha.

Interleukin-1alpha (IL-1alpha) is a multifunctional cytokine that promotes inflammation, tissue remodeling and epithelial hyperplasia. Keratinocytes produce and sequester large amounts of biologically active IL-1alpha which can be released after injury or infection. We show that high level expression of human papillomavirus (HPV) type 16 E6 and E7 oncoproteins enhanced release of IL-1alpha from cultures of normal cervical keratinocytes (relative effectiveness E7 > E6/E7 >> E6 > control). The amount of IL-1alpha released was directly related to the ability of E7 or E6/E7 to stimulate apoptosis. E7 proteins that bound the retinoblastoma protein (Rb) strongly (HPV-16 and -18) induced more IL-1alpha release than those that bound poorly (HPV-6 and an HPV-16 E7 24gly mutant). Furthermore, overexpression of the E2F-1 transcription factor, a downstream target of Rb, induced extensive apoptosis and IL-1alpha release. Apoptosis and IL-1alpha release in response to growth factor removal occurred in part through a p53-independent pathway as coexpression of E6 and downregulation of p53 did not prevent either response. Immunohistochemical analyses showed that IL-1alpha was expressed by keratinocytes in normal cervical epithelia, low and high grade dysplasias, and cervical carcinomas. However, HPV-16 E6/E7 RNA expression and apoptosis increased in parallel in proliferating keratinocytes in severe dysplasias and carcinomas suggesting that IL-1alpha release is associated with progression to high grade disease. Thus, high level expression of the HPV-16 E7 protein sensitizes keratinocytes to apoptosis which results in release of IL-1alpha.

Relationship of stable integration of herpes simplex virus-2 Bg/II N subfragment Xho2 to malignant transformation of human papillomavirus-immortalized cervical keratinocytes.

Transfection of the right end Xho2 subfragment of Bg/II N of herpes simplex virus-2 (HSV-2) into human genital keratinocytes immortalized by human papillomavirus (HPV) type 16 or 18 resulted in invasive and noninvasive indolent cystic squamous carcinomas when cells were injected into immunocompromised mice. Retention and expression of the right end portion of the Bg/II N fragment correlated with malignancy, as the corresponding HSV-2 sequences were integrated and transcribed in the tumorigenic cell lines. HPV-immortalized cells alone were not tumorigenic. In contrast, previous results have shown that using the entire Bg/II N region can malignantly transform HPV-immortalized cells, although HSV2 DNA was not retained. Together, these observations localize the transforming activity of Bg/II N to Xho2 and suggest that the remaining sequences have an inhibitory effect on stable integration. The Xho2 sequence is 2480 bp long and contains an open reading frame (ORF) extending from nucleotides 559 to 1797. The ORF encodes a putative protein of 412-aa with a m.w. of 42-43 kDa and is highly homologous to UL43 of HSV-I. The correlation of tumorigenicity with stable integration and expression of Xho2 DNA in HPV-immortalized cells indicates that HSV-2 should be investigated further for a possible role in cervical cancer.

Expression of 4alpha-carbinolamine dehydratase in human epidermal keratinocytes.

4alpha-Carbinolamine dehydratase is a bifunctional protein involved in the regeneration of tetrahydrobiopterin during the hydroxylation of the aromatic amino acids. It is also a dimerization cofactor of HNF1 and therefore is believed to function as part of the hepatic gene transcription system. In view of the recent discoveries that the distribution and developmental pattern of the dehydratase do not correlate strictly with those of the aromatic amino acid hydroxylases and HNF1, the hypothesis that the dehydratase may have other unknown functions has been put forward. In the present paper, we demonstrate unambiguously that human epidermal keratinocytes express detectable levels of this protein as indicated by enzyme assay, immunoprecipitation, Western blot, and RT-PCR. Its complete coding sequence has been cloned and was found to be identical with the human liver counterpart. The possible function of the dehydratase in skin is discussed.

Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.

Wild-type p53 (wt-p53) negatively controls cell cycle progression after cellular stress mediating either a temporary growth arrest or apoptosis, depending on the cell type and nature of the cellular stress. The aberrant proliferation which is characteristic of tumor cells may be suppressed by exogenous wt-p53 and appears to depend strongly on the level of reexpression. We performed retroviral-mediated gene transfer of wt-p53 into a human squamous cell carcinoma cell line from the head and neck region (A253 cell line) lacking endogenous p53. This allowed us to study the effect of wt-p53 on the malignant phenotype and on the response to the DNA damaging agent ultraviolet B (UVB). Restoration of wt-p53 in malignant keratinocytes suppressed tumorigenicity in nude mice although p53-reconstituted cells eventually formed small tumors with long latency. Cells derived from these tumors showed reduced expression of wt-p53. Exogenous wt-p53 increased baseline mRNA expression of the small proline rich proteins 1 and 2, consistent with a prodifferentiating effect. After exposure to a biological UVB dose, only p53-positive A253 cells underwent an early and transient G1 arrest. Both p53-positive and -negative A253 cells displayed a late G2 delay/arrest. We conclude that reexpression of wt-p53 in squamous cell carcinoma A253 cells decreases their malignant phenotype and reestablishes a G1 checkpoint after UVB.

Altered expression of Erg and Ets-2 transcription factors is associated with genetic changes at 21q22.2-22.3 in immortal and cervical carcinoma cell lines.

Human Papillomavirus (HPV) type 16 is the most frequently detected HPV in cervical cancer. Although epidemiologic and experimental evidence indicates a prominent role for HPV infection in the development of this disease, other factors are also involved. Altered expression of the ets family transcription factors erg and ets-2 was found associated with the development of cervical carcinoma. Overexpression also occurred in a HPV-16-immortalized cervical cell line, CX16-2, which has HPV integrated at a translocation breakpoint t(19;21) involving 21q22.2-22.3, where these genes have been mapped. Six of 10 cervical carcinoma cell lines overexpressed ets-2 RNA suggesting an association of overexpression with cervical cell neoplasia. A clonally related pair of cervical carcinoma cell lines, C-4I and C-4II, showed differential expression of erg and ets-2. C-4I overexpressed ets-2 RNA compared to normal cervical cells and C-4II. C-4II expressed a 5.3 kb erg transcript not seen in C-4I, ectocervical cells or other cervical carcinoma cell lines examined. Pulsed field gel electrophoresis was used to analyse changes in DNA fragments related to structural changes and to construct a physical map encompassing erg and ets-2. Alterations in erg and ets-2 RNA expression in each of three different cell lines examined were associated with translocations. Association between altered expression of erg and ets-2 and altered regional structural suggests that these genes are important targets in cervical carcinogenesis.

Papillomaviruses and potential copathogens.

An in vitro multistage genital epithelial cell model for cervical cancer that parallels the in vivo neoplastic process has been developed using recombinant human papillomavirus (HPV) DNA and genital cells. HPV-16-immortalized genital cells are responsive to the genotoxic action of known chemical carcinogens (polycyclic hydrocarbons, alkylating agents or cigarette smoke condensate), but are not converted to malignancy. Ras oncogene and human herpes virus-2 did convert HPV immortalized cells to malignancy, whereas human herpes virus-6 infection only increased HPV expression. Human immunodeficiency virus did not infect genital cells.

Expression and action of parathyroid hormone-related peptide in human cervical epithelial cells.

Parathyroid hormone-related peptide (PTHRP) expression and activity were analyzed in normal human ectocervical keratinocytes (HCX) and keratinocytes immortalized by transfection with human papillomavirus (HPV) types 16 and 18 DNAs. In normal cells, trans-retinoic acid (RA) and 2.0 mM Ca2+ significantly stimulated PTHRP mRNA expression and secretion and led to a significant reduction in the rate of proliferation. In contrast, the basal level of PTHRP production decreased sharply in confluent HCX, and induction by Ca2+ or exogenous growth factors was reduced or lost. After stable transfection with HPV16 and HPV18 DNAs, we observed a sharp decrease of PTHRP production in high-passage poorly differentiated HCX. Finally, addition of exogenous PTHRP-(1-141) inhibited proliferation of both normal cells and low-passage well-differentiated HPV16 immortalized cells. High-passage poorly differentiated cells were refractory to PTHRP. These results demonstrate that PTHRP production varies greatly with the degree of cell proliferation and differentiation and suggest that this peptide acts as an autocrine negative growth regulator for cervical keratinocytes.