Interferon-alpha elicits downregulation of human papillomavirus 18 mRNA in HeLa cells by selective repression of endogenous viral transcription.

We have reported that interferon-alpha inhibits HPV-18 mRNA in HeLa cells. Here we examine mechanisms by which IFN could modulate HPV expression. In northern blot experiments, we observed that interferon-alpha 2b treatment reduced HPV-18 mRNA levels in a time- and dose-dependent manner, with a maximal effect achieved at 48 h. Simultaneously, induction of 2-5A synthetase mRNA was verified as indicative of IFN action. The IFN regulatory effect on HPV-18 mRNA at 48 h required de novo protein synthesis. We performed run-on experiments to determine whether the IFN regulatory effect was at the transcriptional level. HPV-18 endogenous transcription was repressed using 200 and 1000 IU/ml. Interferon treatment did not affect HPV-18 mRNA stability, at least under our experimental conditions. To verify whether HPV-18 enhancer sequences were involved in the interferon effect, we transfected a construct containing the chloramphenicol acetyltransferase driven by the HPV-18 upstream regulatory region. The enzyme activity was unmodified on human keratinocytes and HeLa cells by interferon exposition. Our data demonstrate that interferon-alpha downregulates HPV-18 mRNA levels on HeLa cells by repressing nascent viral transcripts, possibly through regulatory cellular flanking regions.

Modulation of human papillomavirus type 16 mRNA in cervical invasive carcinoma patients by interferon-alpha therapy.

Mechanisms by which interferon produces papilloma regression remain largely unknown. We analyzed biopsies from three cervical invasive carcinoma patients treated with interferon-alpha (IFN-alpha) administered both topically and i.m. for 15 days. All specimens contained human papillomavirus (HPV-16) DNA as determined by polymerase chain reaction using specific HPV-16 E7 primers. Interestingly, in two patients. HPV-16 mRNA expression was reduced (44% and 67%, respectively) after IFN treatment. Upregulation of 2-5 A synthetase and PKR mRNA levels were indicative of the IFN effect. A larger study should be initiated to see whether IFN-alpha modulates the HPV-16 mRNA levels in tumor biopsies from cervical carcinoma patients.

IFNalpha 2b induces apoptosis and proteasome-mediated degradation of p27Kip1 in a human lung cancer cell line.

IFNs are a family of cytokines involved in antiviral defense, cell growth regulation and immune activation. IFNs either inhibit cell proliferation or control apoptosis depending on factors such as cell type and state of cell differentiation. It is important to determine how IFN-induced gene products interact with other cellular proteins to produce these responses. We have investigated the effect of IFNalpha 2b on a human small cell lung carcinoma (SCLC) cell line H82. We have found that IFNalpha efficiently induces apoptosis in H82 cells. The induction of apoptosis by IFNalpha 2b is accompanied by decreased levels of c-myc and Cdk2. We have also observed that in H82 cells IFNalpha induces downregulation of p27 and this is in contrast to the upregulation of p27 observed in other cell types where IFNs induce cell cycle arrest. IFNalpha-induced downregulation of p27 is due to protein destabilization and can be prevented by the proteasome inhibitor LLnL. The data suggest that in H82 cells, IFNalpha 2b induces degradation of p27Kip1 independently of CDK2 kinase activity and through a ubiquitin or ubiquitin-related pathway and that the degradation of p27Kip1 could be a molecular event of importance for IFN-induced apoptosis in cancer cells.

Human papillomavirus type 16 E7 impairs the activation of the interferon regulatory factor-1.

We had previously observed that HPV-16 E7 disturbs the Guanylate Binding Protein (GBP)-ISRE reporter activation by IFN-gamma thus suggesting an alteration of the IRF-1 function. In this study we examined the mechanism by which E7 affects the IFN-gamma signals driving the activation of gene transcription. Using 14/2 BRK cells containing dexamethasone-inducible HPV-16 E7 gene, we observed a large inhibition of the IRF-1 DNA binding activity upon E7 induction. Concomitantly, there was no significant change in the levels of IRF-1, indicating that this was not due to reduced levels of IRF-1 expression. Likewise, in vitro translated E7 did not affect the IRF-1 DNA binding activity in nuclear extracts derived from IFN-induced cells, thus indicating that the effects of E7 are upstream of IRF-1's binding to its DNA recognition site. Finally, NFkappaB DNA binding activity was also inhibited under conditional expression of E7. These data indicate that HPV-16 E7 inhibits the IRF-1 and NFkappaB function and this could lead to the impairment of the IFN response in HPV-infected cells. Furthermore, the findings suggest that different events of the IFN inducible signal cascade seem to be target for HPV-16 E7.

Detection and typing of human papillomavirus DNA in benign and malignant tumours of laryngeal epithelium.

The role of human papillomaviruses (HPV) in laryngeal squamous cell carcinoma has not yet been established. Thirty-three cases of laryngeal squamous cell carcinoma were analysed for the presence of HPV DNA and compared with 25 cases of normal larynx and 29 cases of laryngeal squamous papilloma in their positivity index. The presence of HPV DNA was analysed by using L1 consensus primers and also by primers specific for the E7 gene of HPV types 16 and 18. Four normal laryngeal samples (16%) were positive for HPV DNA against the 24 samples (82%) (p < 0.001) found for laryngeal papilloma and 16 (48.5%) (p < 0.05) found for laryngeal squamous cell carcinoma. HPV 16 was the type most frequently found in laryngeal carcinoma samples. Our results support an etiologic role for this type of HPV in the pathogenesis of laryngeal carcinoma.

[Expression of interferon alpha receptors in cell lines from human tumors. Relationship with the antiproliferative effect]. / Expresión de receptores para el interferón alfa en líneas celulares procedentes de tumores humanos. Relación con el efecto antiproliferativo.

Alpha Interferon (IFN) membrane receptor expression was studied in four human tumor cell lines from several origins: Burkitt's lymphoma (Daudi cells), cervix adenocarcinoma (HeLa cells), larynx carcinoma (HEp-2 cells) and a grade III-IV glioblastoma (GL-5 cells). The number of receptors per cell expressed in each case did not correlate with the sensitivity of the cytostatic effect of IFN alpha-2b. However, the Scatchard analysis of the affinity of the IFN-receptor complex did correlate with this effect. Daudi cells, which expressed 1927 receptors/cells with a high affinity (Kd = 3.30 x 10(-10) M) were the most sensitive to the antiproliferative effect. Growth inhibition was 30% with only 1.6pg/ml of IFN. HeLa cells expressed a mixture of high (Kd = 3.36 x 10(-10) M) and low (Kd = 3.21 x 10(-9) M) affinity binding sites and were also sensitive to the antiproliferative effect but less than Daudi. HEp-2 and GL-5 cells, on the contrary, were very little sensitive to the antiproliferative effect of IFN and only expressed low affinity binding sites (Kd = 1.20 x 10(-8) M and Kd = 8.33 x 10(-9) M) respectively. On the other hand, IFN alpha-2b binding assays to peripheral blood lymphocytes from 8 healthy individuals were also carried out as normal cells for comparison. The values obtained were 433 +/- 166 receptors per cell and Kd = 3.68 2.66 x 10(-10) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial phenotypic reversion of HeLa cells by long-term interferon-alpha treatment.

Human papillomaviruses (HPV) are associated with malignant cervical neoplasia. Several HPV-related diseases have been shown to be sensitive to interferon (IFN) treatment. HeLa cells contain and express the HPV type 18 genome and were used as a model for the evaluation of the viral expression regulation and the effect on the malignant phenotype during IFN treatment. Cells were treated continuously with 200 IU/ml IFN-alpha 2b or natural leukocyte INF-alpha for six passages (42 days). Some IFN-induced changes were observed: decrease of HPV-18 mRNA expression, changes of cell morphology, and reduction of clonogenicity in soft agar. Tumorigenicity in nude mice was not modified. Other targets of the IFN system were analyzed, and an increase of the 2',5'-oligoadenylate synthetase mRNA level and a down-regulation of type I IFN receptor were found. These results demonstrate that long-term IFN-alpha treatment induces a partial phenotypic reversion of HeLa cells to a more differentiated stage were down-regulation of HPV-18 expression could play a central role. It therefore confirms that the IFN-alpha treatment may be therapeutically useful in cervix cancer produced by HPV-18.

Differential expression of the p27Kip1 mRNA in IFN-sensitive and resistant cell lines.

IFNs arrest the growth of a small cell lung cancer (SCLC) cell line NCI-H82 in the G1 phase but not the growth of the derived cell line NCI-H82R. Progression through the G1 phase is controlled by positive and negative regulatory genes. Oncoviral genes can override this control. In this study, we compared the effects of human IFN alpha 2b on the mRNA levels of the Cdk inhibitor p27Kip1 in NCI-H82, NCI-H82R and HPV 16 E7-transfected NCI-H82 cell lines. Induction of the 2-5 Oligoadenylate synthetase (2-5 OAS) gene was used as a marker of IFN-dependent signal transduction The expression of p27Kip1 mRNA increased at 48 and 72 hr after IFN alpha 2b addition in sensitive cells. In contrast, p27Kip1 mRNA had only slight variations in both the resistant and E7-transfected cells. Interestingly, the E7-transfected NCI-H82 cells became resistant to the IFN alpha 2b anti-proliferative effect. Our results suggest that p27Kip1 could be a key mediator of the IFN alpha 2b-induced growth arrest and that HPV 16 E7 might affect p27Kip1 inducibility, originating IFN alpha 2b-resistant cells.

Activation of the human p27(Kip1) promoter by IFNalpha 2b.

p27(Kip1) is one of the key regulatory proteins in cell cycle through inhibition of pRB phosphorylation by suppression of the activity of several cyclin/Cdk complexes. The expression of p27(Kip1) has been shown to be controlled by a posttranslational mechanism, although vitamin D(3) and neuronal differentiation can also induce its mRNA. Recently, the p27(Kip1) promoter was isolated and sequenced from a human leukocyte genomic library. In this report, we demonstrate that IFNalpha 2b, activates the human p27(Kip1) promoter-driven luciferase reporter gene in transient expression assays in H82 cells. This induction might involve two IRF 1-like binding sites present in the p27(Kip1) promoter. To our knowledge this is the first report on the direct activation of the human p27(Kip1) promoter by IFNalpha 2b.

Molecular analysis of resistance to interferon in patients with laryngeal papillomatosis.

Although interferon (IFN)-alpha has been used successfully as an adjuvant therapy in laryngeal papillomatosis, some patients are resistant to this treatment. In order to know which patients will benefit from the therapy, we have tried to find a relationship between the IFN response and the viral and host parameters in the lesion. Detection of viral type and copy numbers by polymerase chain reaction (PCR) showed that all patients infected with human papillomavirus (HPV)-11 were sensitive to the treatment, in contrast to those infected with HPV-6. These differences could be explained in part by the inability of HPV-11 E7 to inhibit the induction of an IFN-responsive element, whereas HPV-6 E7 almost completely inhibited the activity of this promoter in transient transfection experiments. Local immune status in the lesion showed that all HPV-11-infected patients had detectable levels of interleukin (IL)-15 and IFN-gamma mRNA, in contrast to HPV-6-infected patients, in whom mRNA for these cytokines was almost absent. Viral copy numbers and levels of IL-4 mRNA could not be correlated with IFN response. Only one patient resistant to recombinant IFN-alpha2b and negative for HPV DNA presented high titers of neutralizing anti-IFN-alpha2b antibodies. This patient became sensitive when natural IFN-alpha was administered. These results suggest that response to IFN may be a complex phenomenon resulting from the interaction between viral and host elements.

Impaired angiogenic balance and suppression of tumorigenicity in HeLa cells chronically exposed to interferon-alpha.

We have previously reported that IFNalpha-chronic treatment for 41 days induced a partial phenotype reversion on HeLa cells along with a down-regulation of HPV18 mRNA levels. However, tumorigenicity of these cells in nude mice was unchanged. Interestingly, after 1 year of IFNalpha-chronic exposition, HeLa cells failed to induce s.c. tumors when injected into nude mice. In such experimental conditions both HPV18 DNA integration pattern and viral DNA copy number present in HeLa cells remained intact in the nontumorigenic phenotype cells. As result of the treatment with IFNalpha, HeLa cells rendered more resistant to lysis mediated by activated natural killer cells in vitro. Furthermore, IFNalpha-chronic treatment was able to induce VEGF and decrease bFGF mRNA expression, suggesting a potential effect on the angiogenic behavior of these tumoral cells. Thus, long-term treatment of HeLa cells with IFNalpha can accomplish a reversion of the malignant phenotype by a sequential multistep mechanism, in which the antiangiogenic effect of IFNalpha could be one of the contributing events.

Efectos de los interferpnes alfa y gamma sobre células tumorales procedentes de gliomas humanos / Effect of alpha and gamma interferons on human glyoma cells

Tres cultivos celulares primarios procedentes de explantos de gliomas humanos malignos fueron caracterizados fenotípicamente en cuanto a su morfología, velocidad de crecimiento, capacidad tumorigénica en ratones desnudos y a sus sensibilidades al efecto citostático de los interferones * natural y gamma natural, usando un sistema de clonogenicidad en medio semisólido (Human Tumor Stem Cell Assay). Según el diagnóstico patológico, los cultivos celulares obtenidos resultaron ser un astrocitoma grado III (GL-3) y dos glioblastomas multiformes de grado III-IV (GL-2 y GL-5) con un tiempo de doblaje de 37,37 y 36,6 horas, respectivamente. Los tres cultivos tuvieron capacidad de producir tumores palpables en ratones desnudos. Sin embargo, los tumores de glioblastoma aparecieron a los 6 días después de la inoculación y el tumor de astrocitoma apareció a los 27 días, lo cual puede deberse a un grado mayor de indiferenciación de las células de glioblastoma, Los tres cultivos produjeron colonias en agar semosólido, denotando la pérdida de la dependencia de anclaje para su crecimiento. El mayor efecto en la inhibición del crecimiento de colonias de células tumorales fue producido por la preparación de interferón gamma de origen natural y de una manera dosis dependiente. Por el contrario, los tres cultivos celulares primarios mostraron una sensibilidad limitada a los interferones tipo *, que se manifestó de forma constante y que no excedió del 50 % de inhibición de las colonias de células tumorales en las condiciones seleccionadas. Los resultados indicaron la presencia de células malignas en los cultivos obtenidos, lo cual es un aspecto importante a considerar en los ensayos de sensibilidad al efecto citostático realizados en el trabajo, y por otro lado, estos resultados pudieran sugerir la posibilidad del empleo de la preparación de interferón gamma natural en estos casos, y no así de los interferones de tipo *, como fue corroborado posteriormente