Case-control study of candidate gene methylation and adenomatous polyp formation.

PURPOSE: Colorectal cancer (CRC) is one of the most common and preventable forms of cancer but remains the second leading cause of cancer-related death. Colorectal adenomas are precursor lesions that develop in 70-90 % of CRC cases. Identification of peripheral biomarkers for adenomas would help to enhance screening efforts. This exploratory study examined the methylation status of 20 candidate markers in peripheral blood leukocytes and their association with adenoma formation. METHODS: Patients recruited from a local endoscopy clinic provided informed consent and completed an interview to ascertain demographic, lifestyle, and adenoma risk factors. Cases were individuals with a histopathologically confirmed adenoma, and controls included patients with a normal colonoscopy or those with histopathological findings not requiring heightened surveillance (normal biopsy, hyperplastic polyp). Methylation-specific polymerase chain reaction was used to characterize candidate gene promoter methylation. Odds ratios (ORs) and 95 % confidence intervals (95% CIs) were calculated using unconditional multivariable logistic regression to test the hypothesis that candidate gene methylation differed between cases and controls, after adjustment for confounders. RESULTS: Complete data were available for 107 participants; 36 % had adenomas (men 40 %, women 31 %). Hypomethylation of the MINT1 locus (OR 5.3, 95% CI 1.0-28.2) and the PER1 (OR 2.9, 95% CI 1.1-7.7) and PER3 (OR 11.6, 95% CI 1.6-78.5) clock gene promoters was more common among adenoma cases. While specificity was moderate to high for the three markers (71-97 %), sensitivity was relatively low (18-45 %). CONCLUSION: Follow-up of these epigenetic markers is suggested to further evaluate their utility for adenoma screening or surveillance.

Loss of p53 induces epidermal growth factor receptor promoter activity in normal human keratinocytes.

Overexpression of the epidermal growth factor receptor (EGFR) in human papillomavirus type 16-immortalized human keratinocytes (HKc) is caused by the viral oncoprotein E6, which targets p53 for degradation. We have previously observed that expression of p53 RNAi in normal HKc is associated with an increase in EGFR mRNA and protein. We now report that p53 RNAi induces EGFR promoter activity up to approximately 10-fold in normal HKc, and this effect does not require intact p53 binding sites on the EGFR promoter. Exogenous wild-type p53 inhibits the EGFR promoter at low levels, and activates it at higher concentrations. Yin Yang 1 (YY1), which negatively regulates p53, induces EGFR promoter activity, and this effect is augmented by p53 RNAi. Intact p53 binding sites on the EGFR promoter are not required for activation by YY1. In addition, Sp1 and YY1 synergistically induce the EGFR promoter in normal HKc, indicating that Sp1 may recruit YY1 as a co-activator. Wild-type p53 suppressed Sp1- and YY1-mediated induction of the EGFR promoter. We conclude that acute loss of p53 in normal HKc induces EGFR expression by a mechanism that involves YY1 and Sp1 and does not require p53 binding to the EGFR promoter.

Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry.

Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.

Human papillomavirus type 16 E6 and E7 cooperate to increase epidermal growth factor receptor (EGFR) mRNA levels, overcoming mechanisms by which excessive EGFR signaling shortens the life span of normal human keratinocytes.

Epidermal growth factor receptor (EGFR) levels are dramatically increased in human keratinocytes (HKc) immortalized with full-length human papillomavirus type 16 (HPV16) DNA (HKc/HPV16), but increases in EGFR levels actually precede immortalization. In some normal HKc strains, acute expression of HPV16 E6 (but not HPV16 E5, HPV16 E7, or HPV6 E6) from LXSN retroviral vectors produced an increase in EGFR mRNA levels detectable at 24 h and stable for up to 10 days after infection. However, about one-half of the individual normal HKc strains we analyzed proved unresponsive to E6 induction of EGFR mRNA despite the robust expression of E6 and degradation of p53. E6 responsiveness of normal HKc strains correlated inversely with initial EGFR levels: although HKc strains expressing relatively low basal EGFR levels grew poorly and tolerated the infection protocol with difficulty, they responded to E6 with an increase in EGFR mRNA and protein and with robust proliferation. However, those HKc strains expressing high basal EGFR levels grew well, but did not respond to E6 with increased EGFR levels or with proliferation. Immunostaining of paraffin-embedded foreskin tissue for the EGFR confirmed that there is an intrinsic interindividual variability of EGFR expression in HKC: These results prompted us to investigate the effects of overexpression of the EGFR in normal HKC: Infection of normal HKc with a LXSN retrovirus expressing the full-length human EGFR cDNA resulted in a dramatic reduction in growth rate and a shorter life span. Although acute expression (1-10 days after infection) of HPV16 E7 alone did not induce the EGFR, acute expression of E6 and E7 together increased EGFR levels in normal HKc unresponsive to E6 alone. Also, HKc infected with E7 alone expressed increased EGFR levels at early stages of extended life span (at passage 9 after infection), and HKc immortalized by HPV16 E7 alone expressed EGFR levels comparable with those of E6/E7-immortalized cells. These results support a key role of the EGFR in HPV16-mediated transformation of HKC: In addition, these data show that normal HKc do not tolerate excessive EGFR levels/signaling, and such intolerance must be overcome in order for HKc to become immortalized by HPV16. We conclude that both E6 and E7 contribute to increasing EGFR levels, but with different mechanisms: although E6 can increase EGFR levels, it cannot overcome the resistance of normal HKc to excessive EGFR signaling. On the other hand E7, which alone does not acutely increase EGFR mRNA or protein, allows for EGFR overexpression in normal HKC:

MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products.

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).

Unique carboxyl-terminal sequences of wild type and alternatively spliced variant forms of transforming growth factor-alpha precursors mediate specific interactions with ErbB4 and ErbB2.

We have previously reported that the human transforming growth factor-alpha (TGF-alpha) gene encodes three forms of TGF-alpha precursors, designated wild type (WT), variant I (VaI), and variant II (VaII), derived from alternative splicing. The two carboxyl-terminal valine residues of WT are replaced by 5 (GCRLY) or 4 (ATLG) amino acids in VaI or VaII, respectively. When overexpressed in Chinese hamster ovary (CHO) cells, VaI and ValI, but not WT, support autonomous growth. We detected tyrosine phosphorylation of ErbB2 in the absence of serum, in CHO cells expressing WT, VaI, or VaII, but not in mock transfectants. These observations prompted us to investigate possible interactions between the ErbBs and the TGF-alpha precursors in CHO cells. All TGF-alpha precursors were found to co-immunoprecipitate with the ErbBs, but with different specificity. WT co-immunoprecipitated with ErbB4, but not with ErbB1, ErbB2, or ErbB3. VaI and VaII co-immunoprecipitated with ErbB2, but not with ErbB1, ErbB3, or ErbB4. Confocal fluorescent microscopy analysis demonstrated that WT, VaI, and VaII all distribute equally to the cell surface while, as expected, a WT mutant lacking the two C-terminal valine residues does not. Point and deletion mutants involving the unique carboxyl-terminal residues of WT, VaI and VaII, indicated that the interactions between the three TGF-alpha precursors and the ErbBs were mediated by their carboxyl-terminal regions, which constitute distinct protein-binding motifs. A chimera of the intracellular domain of WT TGF-alpha linked to exogenous transmembrane and extracellular domains retained both the cell surface distribution and the specific interaction with ErbB4 of full-length WT, confirming that this interaction is mediated by the C-terminus of the TGF-alpha precursor. While interactions of WT and variant TGF-alpha with the ErbBs all result in ErbB2 activation, they produce different biological consequences, suggesting that the various TGF-alpha precursors differentially modulate ErbB signaling.

Retinoic acid resistance at late stages of human papillomavirus type 16-mediated transformation of human keratinocytes arises despite intact retinoid signaling and is due to a loss of sensitivity to transforming growth factor-beta.

In our in vitro model of human cell carcinogenesis, normal human foreskin keratinocytes (HKc) transfected with human papillomavirus type 16 DNA (HKc/HPV16) progress toward malignancy through several phenotypically defined and reproducible "steps" that include immortalization, growth factor independence (HKc/GFI), differentiation resistance (HKc/DR), and ultimately malignant conversion. While HKc/HPV16 are very sensitive to growth inhibition by all-trans-retinoic acid (RA) at early passages, they lose their sensitivity to RA during progression in culture. However, gel mobility shift assays using the retinoid response elements DR1 and DR5 showed no changes in binding activity of nuclear extracts obtained from HKc/HPV16 at different stages of in vitro progression. Similarly, Western blot analyses for retinoic acid receptor gamma-1 and the retinoid X receptors failed to reveal any decreases in the levels of these retinoid receptors throughout progression. In addition, luciferase activity driven by the SV40 promoter with a DR5 enhancer element was activated following RA treatment of HKc/DR that were resistant to growth inhibition by RA. Since RA induces transforming growth factor-beta2 (TGF-beta2) in normal HKc and HKc/HPV16, we investigated whether this response changed during progression. Again, RA induced TGF-beta2 mRNA in early and late passage HKc/HPV16, HKc/GFI, and HKc/DR approximately to the same extent, confirming that the RA signaling pathways remained intact during in vitro progression despite the fact that the cells become resistant to growth inhibition by RA. We then investigated the sensitivity of HKc/HPV16 to growth inhibition by TGF-beta. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta1 and TGF-beta2, the cells became increasingly resistant to both TGF-beta isotypes during in vitro progression. In addition, while both RA and TGF-beta produced a decrease in the levels of mRNA for the HPV16 oncogenes E6 and E7 in early passage HKc/HPV16, this effect was also lost at later stages of progression. Finally, blocking anti-TGF-beta antibodies partially prevented RA inhibition of growth and E6/E7 expression in early passage HKc/HPV16. Taken together, these data strongly suggest that inhibition of growth and HPV16 early gene expression in HKc/HPV16 by RA is mediated by TGF-beta and that a loss of RA sensitivity is linked to TGF-beta resistance rather than alterations in RA signaling.

Loss of transforming growth factor-beta (TGF-beta) receptor type I mediates TGF-beta resistance in human papillomavirus type 16-transformed human keratinocytes at late stages of in vitro progression.

Human keratinocytes (HKc) immortalized by human papillomavirus type 16 DNA (HKc/HPV16) progress toward malignancy through growth factor-independent (HKc/GFI) and differentiation-resistant stages (HKc/DR). This progression is associated with a loss of sensitivity to growth inhibition by both all-trans-retinoic acid (RA) and transforming growth factor-beta (TGF-beta). In the accompanying article (Borger et al., 2000, Virology 270, 397-407), we demonstrate that RA resistance in HKc/HPV16 arises despite functional nuclear retinoid receptors and that TGF-beta mediates growth inhibition by RA. To investigate the basis for the loss of TGF-beta sensitivity during in vitro progression of HKc/HPV16, we explored the expression of TGF-beta receptors type I and type II in independently derived HKc/HPV16 lines and their corresponding HKc/GFI and HKc/DR derivatives. While TGF-beta receptor type II mRNA levels were unchanged during progression, mRNA levels for TGF-beta receptor type I decreased dramatically as the cells became TGF-beta resistant. At the HKc/DR stage, loss of TGF-beta receptor type I mRNA, compared to low-passage cells, ranged from 55 to 87% in four HKc/HPV16 lines examined. Immunohistochemistry, using anti-TGF-beta receptor type I antibodies, confirmed a loss of TGF-beta receptor type I expression in HKc/DR. Reintroduction of the TGF-beta-receptor type I into TGF-beta-resistant HKc/DR completely restored growth inhibition by TGF-beta. Southern blot analysis of DNA extracted from normal HKc, HKc/HPV16, and HKc/DR ruled out any gross changes in the TGF-beta receptor type I gene. The activity of the TGF-beta receptor type I promoter, cloned upstream of a luciferase reporter gene, was decreased in HKc/DR, to an extent comparable to the decrease in mRNA levels for the TGF-beta receptor type I. Thus, TGF-beta resistance at late stages of HPV16-mediated transformation of HKc is the result of a loss of expression of TGF-beta receptor type I.

Population-based, case-control study of HER2 genetic polymorphism and breast cancer risk.

BACKGROUND: Alterations of the HER2 (also known as erbB-2 or neu) proto-oncogene have been implicated in the carcinogenesis and prognosis of breast cancer. A polymorphism at codon 655 (GTC/valine to ATC /isoleucine [Val(655)Ile]) in the transmembrane domain-coding region of this gene has been identified and may be associated with the risk of breast cancer. We evaluated this hypothesis in a subgroup of women who participated in a large-scale, population-based, case-control study of breast cancer in Shanghai, China. METHODS: Genomic DNA from 339 patients with breast cancer and 361 healthy control subjects was examined for the Val(655)Ile polymorphism with a polymerase chain reaction-restriction fragment-length polymorphism-based assay. All study subjects completed a structured questionnaire during an in-person interview. All P values are from two-sided tests. RESULTS: We found that 25.1% of the case patients and 21.7% of the control subjects were heterozygous for the Val allele and 3.2% of the case patients and 0. 3% of the control subjects were homozygous for this allele (P =.005). Compared with women with the Ile/Ile genotype, women who had the Ile/Val or Val/Val genotype had an elevated risk of breast cancer (odds ratio [OR] = 1.4; 95% confidence interval [CI] = 1.0-2.0; P =. 05) after adjustment for age, educational level, study period, history of breast fibroadenoma, leisure physical activity, and age at first live birth. The risk was elevated even more among women who were homozygous for the Val allele (OR = 14.1; 95% CI = 1.8-113.4). The association was more pronounced among younger women (45 years). The adjusted OR associated with the Val allele was 1.7 (95% CI = 1.1-2.6) for younger women and 1.0 (95% CI = 0.5-1.9) for older women. CONCLUSIONS: Results of this study suggest that polymorphisms of the HER2 gene may be important susceptibility biomarkers for breast cancer risk, particularly among younger women.

Human keratinocytes and tumor-derived cell lines express alternatively spliced forms of transforming growth factor-alpha mRNA, encoding precursors lacking carboxyl-terminal valine residues.

The human transforming growth factor-alpha (TGF-alpha) gene is thought to contain five introns and six exons, encoding a transmembrane precursor (proTGF-alpha) from which the mature polypeptide is released by proteolytic cleavage. We identified a novel 32-nucleotide exon (exon alpha) within intron 5 and an alternative splice acceptor site in exon 6, splitting exon 6 into two segments: 6A and 6B. Therefore, in addition to wild type (wt) proTGF-alpha mRNA, which skips exon alpha, two novel proTGF-alpha variants are produced: Variant I (VaI), skipping exons alpha and 6A, and Variant II (VaII) which includes exon alpha and skips exon 6A. The only significant difference between variant and wt proTGF-alpha proteins is that the two wt carboxyl-terminal valines are replaced in the variants by five or four other amino acids, respectively. Both variant TGF-alpha mRNAs were readily detected in human keratinocytes and tumor-derived cell lines. Their protein products were cleaved as efficiently as wt TGF-alpha in response to the calcium ionophore A23187. However, both variants (but not wt) reduced serum requirements for proliferation in CHO cells. In addition, VaII-expressing CHO cells (not VaI or wt) formed foci in monolayer cultures. These results suggest that variant TGF-alpha precursors induce autonomous growth.

Comparison of the metabolism of retinol delivered to human keratinocytes either bound to serum retinol-binding protein or added directly to the culture medium.

Retinol (vitamin A) circulates in the blood bound to retinol-binding protein (RBP), which is thought to be responsible for the delivery of the vitamin to target cells, including the basal cells of the skin (keratinocytes). The process by which keratinocytes acquire retinol from RBP remains controversial. A mechanism for retinol delivery to keratinocytes involving cell surface RBP receptors has been proposed, while other studies support an RBP receptor-independent process. To further explore retinol uptake we have used a model system of human foreskin keratinocytes cultured in serum-free media to compare the metabolism of [3H]retinol delivered to the cells either bound to RBP or added directly to the culture medium. The majority of the cell-associated radioactivity found in keratinocytes incubated for 0.5 to 24 h with either free or RBP bound [3H]retinol was present as [3H]retinyl ester irrespective of the mode of delivery. In keratinocytes incubated for 24 h with [3H]retinol added directly to the culture medium or bound to RBP, [3H]retinyl ester comprised 76 and 80%, respectively, of the total cell-associated radioactivity. Also, the relative cellular levels of the different retinyl esters species synthesized by the keratinocytes were the same whether the [3H]-retinol was delivered free or bound to RBP. Finally, the kinetics of loss (turnover) of cell-associated [3H]-retinol and [3H]retinyl esters from keratinocytes pre-labeled with [3H]retinol delivered free or bound to RBP was the same. Overall, this study demonstrates that the rate and extent of retinol esterification by keratinocytes and the types of esters synthesized are the same whether the vitamin is delivered to the cells free or bound to RBP and argues against RBP receptor-mediated delivery of retinol to specific sites on the plasma membrane that influence overall retinol metabolism.

Glucocorticoids stimulate growth of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes and support HPV16-mediated immortalization without affecting the levels of HPV16 E6/E7 mRNA.

We investigated the effects of the glucocorticoids hydrocortisone and dexamethasone on human papillomavirus type 16 (HPV16)-mediated human cell carcinogenesis using normal human keratinocytes (HKc) and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16). Normal HKc did not require glucocorticoids for proliferation. In contrast, growth of early passage HKc/HPV16 strictly required these hormones, although glucocorticoid dependence became less stringent during in vitro progression. Glucocorticoid dependence was acquired by HKc early after immortalization with HPV16 DNA, and glucocorticoids were required for efficient HKc immortalization. However, treatment of HKc/HPV16 with hydrocortisone or dexamethasone did not increase the steady-state levels of HPV16 E6/E7 mRNA or protein. Firefly luciferase activity expressed under the control of the HPV16 upstream regulatory region and P97 promoter increased by about fourfold following dexamethasone treatment of HeLa, but only twofold in HKc/HPV16, and less than twofold in SiHa. However, all of these cell lines expressed sufficient endogenous glucocorticoid receptors to allow for a dexamethasone response of the mouse mammary tumor virus promoter. These results indicate that mechanisms other than a direct influence by glucocorticoids on HPV16 early gene expression may contribute to the striking biological effects of these steroids on HPV16-mediated human cell carcinogenesis.

Evaluation of transdermal penetration enhancers using a novel skin alternative.

A novel alternative to animal skin models was developed in order to aid in the screening of transdermal penetration enhancers. The skin alternative consists of a dermal layer containing human fibroblasts dispersed in a collagen matrix and an epidermal layer of differentiated and stratified human keratinocytes. Skin alternatives were placed in modified Franz diffusion cells (receptor volume 12 mL, donor area 3.14 cm2, n = 5) and enhancer solution (0.4 M in propylene glycol (PG)) was applied. Following 1 h of pretreatment, 10 microL of saturated hydrocortisone (HC) solution in PG was applied, and the cells were occluded with Parafilm. Samples were removed from the receptor compartment over 24 h, replaced with fresh receptor solution, and analyzed for steroid content using HPLC. Skin HC content was also determined. Receptor concentration at 24 h (Q24) for full-thickness skin alternative (control) was 28.6 +/- 13.7 microM and permeability (P) was 8.3 x 10(-4) +/- 5.5 x 10(-4) cm h-1. Azone (1) produced a Q24 of 105.0 +/- 36.1 microM and a P of 11.3 x 10(-4) +/- 1.8 x 10(-4) cm h-1, while the novel penetration enhancer 1-dodecyl-2-pyrrolidinone (2) produced a Q24 of 164.8 +/- 61.2 microM and a P of 33.3 x 10(-4) +/- 6.6 x 10(-4) cm h-1. Compound 3 produced the highest values for all permeability parameters tested with a P of 48.0 +/- 36.8 cm h-1 and a Q24 of 186.1 +/- 45.1 microM. When compared to the control, compound 1 gave an enhancement ratio (ER) of 3.7 for Q24 and 1.4 for P. Compound 2 gives an ER 5.8 for Q24 and 4.0 for P. These enhancement ratios are similar to those found using HC and human skin.

Uptake and metabolism of [3H]retinoic acid delivered to human foreskin keratinocytes either bound to serum albumin or added directly to the culture medium.

Retinoic acid (RA), a potent modulator of cell proliferation and differentiation is present in plasma bound to serum albumin. The biologic significance or source of plasma RA is not clear. Although most cellular RA is believed to be made in situ via the oxidation of retinol, plasma RA could potentially provide target cells with a source of preformed RA. To investigate RA uptake, we have used a model system of human foreskin keratinocytes (HKc) cultured in serum-free media to compare the uptake and metabolism of [3H]RA added directly to the culture medium in ethanol to that delivered bound to bovine serum albumin (BSA). [3H]RA added directly to the culture medium was rapidly taken up by HKc during the first 10 min of incubation (25-35% of the applied RA), no further accumulation occurred between 10 min and 90 min, and then cell-associated radioactivity rapidly decreased to about 3-5% of the applied dose by 12 h. In contrast, when [3H]RA was delivered to HKc bound to BSA, total cell-associated radioactivity reached about 2.5% of the applied dose by 5 min, increased to 3-5% of the applied radioactivity by 1 h, and no further accumulation or loss occurred over the next 23 h. The uptake by HKc of [3H]RA delivered bound to BSA or added directly to the culture medium was not influenced by pre-treatment of the cells for 72 h with unlabeled RA or by excess unlabeled RA added at the time of uptake. Analysis of the cells and media by high-performance liquid chromatography for RA metabolites found that [3H]RA added directly to the medium is rapidly converted by HKc to polar compounds that are subsequently excreted back into the medium. Also, RA added directly to the medium was susceptible to degradation in the absence of cells. In marked contrast, [3H]RA added to the media bound to BSA was much less susceptible to degradation in the absence of cells, and few [3H]RA metabolites were found in the media even after exposure to HKc for 24 h. The binding of RA to albumin clearly protects RA from conversion to polar metabolites, and also provides for a controlled delivery of RA from the aqueous extracellular environment to the cell surface.

Progressive loss of sensitivity to growth control by retinoic acid and transforming growth factor-beta at late stages of human papillomavirus type 16-initiated transformation of human keratinocytes.

Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor beta-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human keratinocytes (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/HPV16, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, growth factor independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium- induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to growth control by all-trans-retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to growth inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. Growth inhibition by RA may be mediated through the cytokine transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose-and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-beta. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-beta 1 and TGF-beta 2 expression about 3- and 50-fold, respectively. We next studied the effect of TGF-beta 1 and TGF-beta 2 on the proliferation of early to late passage HKc/HPVa6, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta 1 and TGF-beta 2, the cells became increasingly resistant to TGF-beta during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-beta 1 or TGF-beta 2 treatment. Overall, loss of growth inhibition by RA parallels loss of TGF-beta sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased levels and constitutive tyrosine phosphorylation of the epidermal growth factor receptor contribute to autonomous growth of human papillomavirus type 16 immortalized human keratinocytes.

Transfection of individual normal human foreskin keratinocyte (HKc) strains with human papillomavirus type 16 (HPV16) DNA results in the establishment of immortalized cell lines (HKc/HPV16) which, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation in serum-free media. However, sublines which proliferate in serum-free media in the absence of EGF and BPE can be reproducibly established from individual HKc/HPV16 lines, following selection in serum-free media lacking EGF and BPE. The growth factor-independent sublines (HKc/GFI) proliferate in the absence of EGF and BPE at the same rate and to the same extent as in medium supplemented with these growth factors, whereas the parental HKc/HPV16 lines proliferate poorly in the absence of EGF and BPE. As a first approach to understanding the molecular basis by which HKc/GFI have lost their requirement for EGF, we compared EGF uptake and EGF receptor (EGFR) numbers in normal HKc, HKc/HPV16, and HKc/GFI. HKc/GFI exhibit increased EGF uptake and increased EGFR numbers compared to HKc/HPV16. A neutralizing antibody against the extracellular domain of the EGFR dramatically inhibited clonal growth of HKc/GFI, indicating that signaling through the EGFR must be important for the ability of HKc/GFI to proliferate in the absence of EGF. In addition, while in the absence of EGF normal HKc and HKc/HPV16 exhibited no detectable EGFR tyrosine phosphorylation, the EGFRs in HKc/GFI were tyrosine phosphorylated in the absence of EGF and hyperphosphorylated in the presence of EGF. Although an anti-TGF-alpha antibody inhibited the growth of HKc/GFI, we unexpectedly found that HKc/GFI and HKc/HPV16 secreted comparable and extremely low amounts of TGF-alpha (4 to 9 pg/10(6) cells per 24 h); about 100- to 250-fold less than normal HKc (1018 pg/10(6) cells per 24 h). No other ligands for the EGFR were detected in media conditioned by normal HKc, HKc/HPV16, or HKc/GFI. Thus, while overexpression and constitutive activation of the EGFR appear to be important features of HKc/GFI, enhanced secretion of TGF-alpha or other ligands for the EGFR does not explain the proliferation of HKc/GFI in the absence of EGF and BPE.

Retinoic acid suppresses human papillomavirus type 16 (HPV16)-mediated transformation of human keratinocytes and inhibits the expression of the HPV16 oncogenes.

We have used a model system of normal HKc and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16) to investigate the effect of RA on the growth of HKc/HPV16 and the expression of the HPV16 oncogenes E6 and E7. These studies found that HKc/HPV16 are about 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. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Northern blot analysis of mRNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 oncogenes E6 and E7 as well as the early ORFs E2 and E5 is substantially reduced following RA treatment. In addition, protein levels of E6 and E7, as measured by immunofluorescence (E6 and E7) and Western blot (E7) are also decreased by RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. The RA treatment (1 nM) of normal HKc, during or immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of dietary retinoids in the chemoprevention of HPV-induced cancers.

Retinoic acid inhibition of human papillomavirus type 16-mediated transformation of human keratinocytes.

We previously reported that human keratinocytes (HKc) immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16) are more sensitive than normal HKc to growth inhibition by retinoic acid (RA), and that RA treatment of HKc/HPV16 inhibits HPV16 E6/E7 mRNA expression (L. Pirisi et al., Cancer Res., 52: 187-193, 1992). We now demonstrate that HPV16 E2 and E5 mRNAs are also decreased by RA treatment of HKc/HPV16, indicating a general inhibition by RA on the expression of HPV16 early genes. In addition, protein levels of E6 and E7, as measured by immunofluorescence, are also decreased in a dose-dependent manner following RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. RA treatment (1 nM) of normal HKc, immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of RA in the chemo-prevention of human papillomavirus-induced cancers.

A comparison of the uptake, metabolism and biologic effects of retinol delivered to human keratinocytes either free or bound to serum retinol-binding protein.

In response to body demands, retinol (ROL) is secreted from the liver into the circulation bound to serum retinol-binding protein (RBP). The mechanism by which ROL is transferred from RBP to target cells remains controversial. To study ROL delivery, we have used a model system of cultured human foreskin keratinocytes (HKc) to compare the uptake, metabolism and biologic effects of ROL added either directly to the medium or bound to RBP. [3H]ROL added directly to the medium was rapidly taken up by HKc, and maximal accumulation of [3H]ROL occurred by 3 h. In contrast, [3H]ROL delivered bound to RBP was taken up very slowly but at a linear rate for at least 72 h. Several experimental approaches indicated that ROL uptake from RBP was not mediated by a cell surface receptor for RBP. The rate and extent of [3H]ROL metabolism to retinyl esters was the same whether the ROL was added directly to the medium or bound to RBP. In addition, several biologic responses by HKc to ROL showed the same dose response curves whether the ROL was added directly to the medium or bound to RBP. Overall, the results support a model of ROL uptake from RBP in which the vitamin is first slowly released from RBP into the aqueous phase and then becomes cell associated. In this manner, the cells are provided with a slow but constant supply of ROL and are protected from the rapid and potentially toxic accumulation of ROL that occurs in the absence of RBP.

Retinoic acid induces secretion of latent transforming growth factor beta 1 and beta 2 in normal and human papillomavirus type 16-immortalized human keratinocytes.

Similar cellular responses are elicited by retinoic acid (RA) and transforming growth factor beta (TGF-beta). We investigated the ability of RA to modulate the production of TGF-beta in normal human keratinocytes (HKc) and HKc lines immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16). RA treatment of both normal HKc and HKc/HPV16 resulted in a 2-3-fold induction in secreted levels of latent TGF-beta. The induction in TGF-beta secretion by RA was dose dependent, with significant increases observed with RA concentrations as low as 1-10 nM, and time dependent, with maximal induction occurring about 3 days after initiation of RA exposure. In addition, RA induced intracellular levels of TGF-beta almost 5-fold. Sandwich enzyme-linked immunosorbent assays were used to specifically quantify TGF-beta 1 and TGF-beta 2 secreted by normal HKc and HKc/HPV16 cultured in the absence or presence of RA. RA increased the secreted levels of latent TGF-beta 1 and TGF-beta 2 an average of 2- and 5-fold, respectively, with no major differences in the fold induction between normal HKc and HKc/HPV16. Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced specific transcripts for TGF-beta 1 and TGF-beta 2 about 3- and 50-fold, respectively. RA treatment of HKc had no significant effect on the binding affinity of TGF-beta for its receptors or receptor number. Normal HKc and HKc/HPV16 displayed similar dose-dependent inhibition of proliferation by TGF-beta 1. These studies indicate that RA may regulate growth control in both normal HKc and HKc/HPV16 by enhancing TGF-beta 1 and TGF-beta 2 production, which, after activation at the cell surface, could inhibit cellular proliferation in an autocrine and/or paracrine manner.