TGF-beta 1 and skin carcinogenesis: antiproliferative effect in vitro and TGF-beta 1 mRNA expression during epidermal hyperproliferation and multistage tumorigenesis.

The role of transforming growth factor-beta 1 (TGF-beta 1) in multisage carcinogenesis in mouse skin was assessed by studying its growth inhibitory effects on nontumorigenic and tumorigenic keratinocytes and by examining its mRNA expression in vitro and during epidermal hyperproliferation and multistage carcinogenesis. While growth of primary basal keratinocytes was inhibited by TGF-beta 1 in doses as low as 0.1 ng/mL, the immortal keratinocyte line MCA/3D ("putatively initiated" cells) responded to TGF-beta 1 with slightly reduced sensitivity, and the papilloma-producing keratinocyte line 308 was considerably less sensitive. In contrast, the squamous carcinoma cell line Carc B was completely nonresponsive, and two other tumorigenic cell lines (PDV and PDVC57) were sensitive to growth inhibition by TGF-beta 1. Steady-state levels of TGF-beta 1 mRNA were high in all the malignant cell lines and in line 308 papilloma cells, but low in primary basal cells and in the nontumorigenic keratinocyte lines V2, Reb1, and MCA/3D. Our in vivo studies showed that tumor promoters, but not mitogenic or weak hyperplasiogenic agents, were able to induce transient expression of TGF-beta 1 mRNA in mouse epidermis. A constitutive overexpression of TGF-beta 1 mRNA was observed in malignant carcinomas but not in the benign premalignant lesions, indicating that overexpression may be associated with malignant progression.     

Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalized human cervical epithelial cells.

Keratin expression in human cervical squamous cell carcinoma (SCC) lines differed significantly from both normal and human papillomavirus (HPV) immortalized exocervical cells. Keratin 14 (K14) expression, determined by protein synthesis and mRNA levels, was dramatically down-regulated in the cervical SCC lines while keratin 5 (K5) expression was not. K14 expression was similarly down-regulated in an HPV-16 immortalized cervical cell line after tumorigenic transformation with recombinant v-Ha-ras DNA. Cultures derived from nude mouse tumor explants also exhibited an altered keratin profile and the levels of K14 protein synthesis, as well as K14 mRNA, were not detectable. In both cases K5 protein synthesis was not significantly down-regulated. In addition, neoplastic cervical SCC lines exhibited up-regulation of keratins 7, 8, 13, and 19, combined with slight down-regulation of keratins 6 and 16. Epidermal keratinocytes responded in a different manner to exocervical cells. Transfection of human papillomavirus-immortalized epidermal keratinocytes with the BglII N fragment of herpes simplex virus 2 produced a neoplastic cell line, but K5 and K14 expression remained unchanged. Thus, neoplastic transformation of human exocervical cells, both in vivo (spontaneous cervical SCC) and in vitro (HPV-16- and v-Ha-ras-induced cervical SCC), is accompanied by characteristic changes in keratin expression. The specific down-regulation of K14 in these tumorigenic cervical cells, in the absence of significant changes in the expression of K5, implies that the normal coordinate regulation of K5 and K14 gene expression has been uncoupled.     

Malignant human papillomavirus type 16-transformed human keratinocytes exhibit altered expression of extracellular matrix glycoproteins.

We found that keratinocytes immortalized with human papillomavirus (HPV) type 16 DNA and malignantly converted by H-ras transfection (HPK-1A/ras) exhibit an enhanced ability to synthesize a fibronectin-containing extracellular matrix. Gene expression of fibronectin and thrombospondin was increased in tumorigenic keratinocytes compared to control and immortalized keratinocytes, 6- and 9-fold, respectively. Increased production of soluble and cell surface-associated fibronectin was not specific for HPV 16 transformed keratinocytes. Ad12-SV40-immortalized keratinocytes malignantly converted by H-ras transfection (RHEK-1/ras) also exhibited enhanced expression of fibronectin and thrombospondin, as well as pro-alpha 1 type I collagen. Steady state mRNA levels for autocrine growth-regulatory factors, transforming growth factors alpha and beta 1, were increased in Ad12-SV40 but not HPV 16-transformed human keratinocytes. We then determined whether increased production of fibronectin was associated with aberrant differentiation of transformed keratinocytes. Less than 10% of the HPV 16-transformed cells produced cornified envelopes after suspension-induced differentiation compared to 70% of normal keratinocytes. However, immortalization by HPV 16 DNA was sufficient to confer a differentiation-defective phenotype. Both involucrin mRNA and protein levels were decreased 8-fold in HPV 16-immortalized keratinocytes compared to normal cells and malignant conversion further attenuated involucrin levels. These studies demonstrate that aberrant differentiation is an early event in the transformation of the human keratinocytes and is not the result of enhanced expression of the extracellular matrix proteins. Unlike transformed fibroblastic cell types, up-regulation of fibronectin gene expression and matrix formation is a consistent characteristic of malignantly converted human keratinocytes.     

Increased sensitivity of human keratinocytes immortalized by human papillomavirus type 16 DNA to growth control by retinoids.

Human papillomavirus (HPV) type 16 (HPV16) is associated with a large percentage of cervical malignancies, and HPV16 DNA can immortalize human keratinocytes in vitro. The transforming ability of the virus resides primarily in the open reading frames E6 and E7. Retinoids are potent modulators of growth and differentiation of keratinocytes and have been shown to reverse cervical lesions resulting from HPV infection. We compared the sensitivity of normal human foreskin keratinocytes (HKc) and four immortalized HKc lines, independently obtained by transfection of different normal HKc strains with HPV16 DNA (HKc/HPV16), to growth control by retinoic acid (RA). All the HKc/HPV16 lines were 10- to 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc, while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. In addition, HKc/HPV16 lines were more sensitive than normal HKc to modulation of keratin expression by RA and retinol. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Dot blot analysis of RNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 open reading frames E6 and E7 is reduced 2- to 4-fold by RA. In addition, Northern blot analysis demonstrated that RA inhibition of E6 and E7 expression was both dose and time dependent. Overall, these results suggest that the increased sensitivity of the HKc/HPV16 lines to growth control by RA may be mediated by an inhibition of the expression of HPV16 gene products which are required for the maintenance of continuous growth.     

In vivo cytokeratin-expression pattern of stratified squamous epithelium from human papillomavirus-type-16–immortalized ectocervical and foreskin keratinocytes

The association between human papillomavirus (HPV) type 16 and cervical squamous-cell carcinoma has been well documented, and HPV 16 DNA has been shown to immortalize human genital keratinocytes in vitro. Using a panel of cytokera-tin(CK)-specific antibodies, we examined the CK expression pattern, an important characteristic of epithelia, of the stratified squamous epithelium reconstructed in vivo from monolayer cultures of 2 human ectocervical and 3 human foreskin keratino-cyte cell lines immortalized by HPV 16 DNA. Whereas the abnormal epithelium formed from these grafts presented certain aspects of mature terminal differentiation, such as morphology and expression of CKIO/II, the expression patterns for CK19, and especially CK13, were more obviously abnormal. CK18 expression was not detectable in any of the epithelia formed from the 5 cell lines. In contrast, for an HPV-16–immortalized endocervical cell line and the CaSki cervicalcarcinoma cell line there was expression of CK18. Our results indicate that HPV-16–induced immortalization of keratinocytes is associated with disruption of the normal CK expression pattern in stratified squamous epithelium and that expression of particular CKs can be differentially disrupted.     

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.     

Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer

Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.     

Effect of human papillomavirus 16 oncoproteins on oncostatin M upregulation in oral squamous cell carcinoma

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.     

Transforming growth factor beta1 promotes chromosomal instability in human papillomavirus 16 E6E7-infected cervical epithelial cells

Uterine cervical cancer, the second most frequently occurring cancer in women worldwide, is tightly associated with the expression of high-risk human papillomavirus [mainly human papillomavirus (HPV)-16 and HPV18] oncogenes E6 and E7 and characteristically exhibits chromosomal instability. However, the mechanisms underlying chromosomal instability in cervical cancer are still not fully understood. In this study, we observed that two of three human cervical epithelial cell lines expressing HPV16 E6E7 became immortalized without extensive chromosomal instability and crisis. The introduction of transforming growth factor (TGF)-beta1, a multiple functional cytokine/growth factor, in the culture medium induced crisis, which was associated with massive chromosomal end-to-end fusions and other structural aberrations. The distributions of structural aberrations on individual chromosomes were significantly correlated with the profiles of telomere signal-free ends. The immortalized cells that emerged from the TGF-beta1-induced crisis showed multiple clonal structural aberrations that were not observed in cells without TGF-beta1 treatment. Overexpression of the catalytic subunit of telomerase (hTERT) abolished the effects of TGF-beta1 on chromosomal instability. Interestingly, another HPV16 E6E7-expressing cervical cell line that experienced crisis and telomere dysfunction under ordinary culture condition had a higher level of autocrine TGF-beta1 production than the other two crisis-free immortalized cell lines. Blocking the TGF-beta1 pathway by an inhibitor of TGF-beta1 receptor type I prevented the crisis and telomere-mediated chromosomal instability. In addition, more dramatic telomere shortening was observed in cervical intraepithelial neoplasias having higher expression of TGF-beta1 in vivo. These results together suggest an important role of TGF-beta1 in the early process of cervical carcinogenesis.     

转化生长因子β1正反义基因对人肺巨细胞癌细胞体内外增殖的影响

探讨人转化生长因子β１（ＴＧＦβ１）正反义基因对人肺巨细胞癌细胞（ＰＬＡ－８０１Ｄ）增殖的影响及调控。方法通过基因转染方法,将ＴＧＦβ１正、反义基因分别导入ＰＬＡ－８０１Ｄ细胞,观察转染和未转染细胞体内外增殖变化、ｎｍ２３基因和ｃ－ｍｙｃ基因表达以及细胞凋亡特征。结果与未转染细胞比较,正义ＴＧＦβ１基因转染细胞培养上清中ＴＧＦβ１活性增加,体外生长速度减慢,软琼脂细胞培养集落形成能力降低,裸鼠体内成瘤率下降约４７％,肿瘤的转移受到抑制。其机制可能与正义ＴＧＦβ１基因转染细胞ｎｍ２３基因表达增加,ｃ－ｍｙｃ基因表达减少以及诱发细胞凋亡有关。而反义ＴＧＦβ１基因转染细胞则出现相反结果。结论ＴＧＦβ１对ＰＬＡ－８０１Ｄ细胞的体内外生长具有负调控作用     

Characterization of normal human exocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18 DNA

An in vitro system for studying the interaction between human papillomavirus (HPV) 16 and 18 recombinant DNA and normal human exocervical epithelial cells is described. Eight HPV-immortalized human exocervical epithelial cell lines were established; all the lines contained either integrated HPV16 or 18 sequences and expressed HPV mRNAs. Thus, integration and expression appear to be required for immortalization. Immortalized cells (greater than 200 population doublings to date) divided rapidly (doubling time of 30 to 46 h) and morphologically resembled primary cultures of normal human exocervical epithelial cells. They expressed a keratin pattern consistent with their origin from exocervical epithelium. When cultured at high density or in the presence of serum they terminally differentiated. Sublines resistant to terminal differentiation were selected by growth in serum-supplemented medium. Keratin pattern changes suggest they have some properties in common with cervical squamous carcinoma cells. However, HPV-immortalized cell lines were not tumorgenic in nude mice. Thus, HPV16/18 is not carcinogenic by itself. These cell lines represent an appropriate model for studying factors that regulate HPV gene expression in normal cervical epithelial cells and examining the influence of cocarcinogens on neoplastic progression.     

Decreased expression of human papillomavirus E2 protein and transforming growth factor-beta1 in human cervical neoplasia as an early marker in carcinogenesis

BACKGROUND AND OBJECTIVES: Human papillomavirus (HPV) is thought to be one of the possible causative factors in cervical carcinogenesis, and cervical carcinoma cells are refractory to tumor transforming growth factor (TGF)-beta1. The purpose of this study is to investigate the possible cause-effect association between HPV and TGF-beta1 during cervical tumorigenesis. METHODS: We assessed the expression of HPV capsid proteins, HPV-16 E7, HPV-16 E2 (C and N terminals), TGF-beta1, and their receptors TGF-beta RI and RII by immunohistochemistry in 48 paraffin-embedded blocks of tumor tissue derived from patients of cervical neoplasia. RESULTS: Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, to microinvasive carcinoma (P < 0.05). Levels of TGF-betaRI and TGFbeta-RII stayed the same in all cases. HPV was found in 89.6% of the studied sections, and cervical lesions without HPV infection expressed significantly less TGF-beta1 (P < 0.05). By comparing the expression pattern of TGF-beta1 and HPV in the neoplastic cells with that of normal cervical epithelium in each section, we found loss of HPV-16 E2 higher in CIN3 (15/24) than in CIN1 or CIN2 (3/7), and there is a significant trend that loss of HPV-16 E2 expression correlated with a >50% loss of TGF-beta1 at the lesion site (P < 0.05). CONCLUSIONS: Our result showed co-suppression of HPV and TGF-beta1 expression during progression of cervical squamous cell cancer. Using antibody against HPV-16 E2 may be an auxiliary tool for the investigation of cervical tumor progression.     

Increased tumorigenicity of human keratinocytes harboring human papillomavirus type 16 is associated with resistance to endogenous tumor necrosis factor-α-mediated growth limitation

The aim of this study was to evaluate the relationship between tumorigenicity of cell sublines derived from weakly tumorigenic SKv-e and SKv-I keratinocytes harboring human papillomavirus type 16 (HPV 16) and their susceptibility to autocrine growth limitation mediated by tumor necrosis factor-α (TNF-α). These sublines displayed different in vitro proliferative potential which correlated with tumorigenicity in nu/nu mice. Recombinant TNF-α inhibited in vitro growth of weakly tumorigenic parental SKv cell lines while it did not affect proliferation of their respective highly tumorigenic sublines. Resistance to TNF-α correlated with both increased in vitro proliferation and tumorigenicity. Anti-TNF-α antibodies (Ab) significantly increased in vitro proliferation of weakly tumorigenic parental SKv cells up to the levels of their highly tumorigenic sublines. Growth of highly tumorigenic SKv cells was not affected. On the other hand, proliferation of SKv cells was affected neither by transforming growth factor-β (TGF-β) nor by anti-TGF-β Ab. All SKv cell sublines tested spontaneously released TNF-α, as evaluated by a specific radioimmunoassay; however, the levels of the endogenous cytokine were not related to their proliferative potential and tumorigenicity. An increased resistance to the anti-proliferative effect of TNF-α may be associated with decreased expression of TNF-α receptors (TNF-αR) inasmuch as evaluation of ¹²⁵ I-TNF-α binding and Northern-blot analysis of TNF-αR-specific mRNA showed that highly tumorigenic SKv cell sublines expressed significantly lower numbers of TNF-αR than their respective parental cells. These results show that an increased tumorigenicity of HPV 16-harboring SKv keratinocytes may be, at least partially, due to escape from autocrine TNF-α-mediated growth limitation.