Neoadjuvant Endocrine Therapy Versus Neoadjuvant Chemotherapy in Node-Positive Invasive Lobular Carcinoma.

BACKGROUND: Neoadjuvant chemotherapy (NACT) is often recommended for patients with node-positive invasive lobular carcinoma (ILC) despite unclear benefit in this largely hormone receptor-positive (HR+) group. We sought to compare overall survival (OS) between patients with node-positive ILC who received neoadjuvant endocrine therapy (NET) and those who received NACT. METHODS: Women with cT1-4c, cN1-3 HR+ ILC in the National Cancer Data Base (2004-2014) who underwent surgery following neoadjuvant therapy were identified. Kaplan-Meier curves and Cox proportional hazards modeling were used to estimate unadjusted and adjusted overall survival (OS), respectively. RESULTS: Of the 5942 patients in the cohort, 855 received NET and 5087 received NACT. NET recipients were older (70 vs. 54 years) and had more comorbidities (Charlson-Deyo score ≥ 1: 21.1% vs. 11.5%), lower cT classification (cT3-4: 44.2% vs. 51.0%), lower rates of mastectomy (72.5% vs. 82.2%), lower rates of pathologic complete response (0% vs. 2.5%), and lower rates of postlumpectomy (73.2% vs. 91.0%) and postmastectomy (60.0% vs. 80.8%) radiation versus NACT recipients (all p < 0.001). NACT recipients had higher unadjusted 10-year OS versus NET recipients (57.9% vs. 36.0%), but after adjustment, there was no significant difference in OS between the two groups (p = 0.10). CONCLUSIONS: Patients with node-positive ILC who received NET presented with smaller tumors, older age, and greater burden of comorbidities versus NACT recipients but had similar adjusted OS. While there is evidence from clinical trials supporting efficacy of NET in HR+ breast cancer, our findings suggest the need for further, histology-specific investigation regarding the optimal inclusion and sequence of endocrine therapy and chemotherapy in ILC.

Long-term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets.

Pig islets are an alternative source for islet transplantation to treat type 1 diabetes (T1D), but reproducible curative potential in the pig-to-nonhuman primate (NHP) model has not been demonstrated. Here, we report that pig islet grafts survived and maintained normoglycemia for >6 months in four of five consecutive immunosuppressed NHPs. Pig islets were isolated from designated pathogen-free (DPF) miniature pigs and infused intraportally into streptozotocin-induced diabetic rhesus monkeys under pretreatment with cobra venom factor (CVF), anti-thymocyte globulin (ATG) induction and maintenance with anti-CD154 monoclonal antibody and low-dose sirolimus. Ex vivo expanded autologous regulatory T cells were adoptively transferred in three recipients. Blood glucose levels were promptly normalized in all five monkeys and normoglycemia (90-110 mg/dL) was maintained for >6 months in four cases, the longest currently up to 603 days. Intravenous glucose tolerance tests during the follow-up period showed excellent glucose disposal capacity and porcine C-peptide responses. Adoptive transfer of autologous regulatory T cells was likely to be associated with more stable and durable normoglycemia. Importantly, the recipients showed no serious adverse effects. Taken together, our results confirm the clinical feasibility of pig islet transplantation to treat T1D patients without the need for excessive immunosuppressive therapy.

Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration.

Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.

Canonical Wnt signalling activates TAZ through PP1A during osteogenic differentiation.

TAZ, a transcriptional modulator, has a key role in cell proliferation, differentiation and stem cell self-renewal. TAZ activity is regulated by several signalling pathways, including Hippo, GPCR and Wnt signalling, but the regulatory mechanisms of TAZ activation are not yet clearly understood. In this report, we show that TAZ is regulated by canonical Wnt signalling during osteogenic differentiation. Wnt3a increases TAZ expression and an inhibitor of GSK3ß, a downstream effector of Wnt signalling, induces TAZ. Wnt3a facilitates the dephosphorylation of TAZ, which stabilises TAZ and prevents it from binding 14-3-3 proteins, thus inducing the nuclear localisation of TAZ. Dephosphorylation of TAZ occurs via PP1A, and depletion of PP1A blocks Wnt3a-induced TAZ stabilisation. Wnt3a-induced TAZ activates osteoblastic differentiation and siRNA-induced TAZ depletion decreases Wnt3a-induced osteoblast differentiation. Taken together, these results show that TAZ mediates Wnt3a-stimulated osteogenic differentiation through PP1A, suggesting that the Wnt signal regulates the Hippo pathway.

TM-25659 enhances osteogenic differentiation and suppresses adipogenic differentiation by modulating the transcriptional co-activator TAZ.

BACKGROUND AND PURPOSE: The transcriptional co-activator with PDZ-binding motif (TAZ) is characterized as a transcriptional modulator of mesenchymal stem cell differentiation into osteoblasts and adipocytes. Moreover, increased TAZ activity in the nucleus enhances osteoblast differentiation and suppresses adipocyte development by interacting with runt-related transcription factor 2 (RUNX2) and PPARγ, respectively. Therefore, it would be of interest to identify low MW compounds that modulate nuclear TAZ activity. EXPERIMENTAL APPROACH: High-throughput screening was performed using a library of low MW compounds in order to identify TAZ modulators that enhance nuclear TAZ localization. The effects and molecular mechanisms of a TAZ modulator have been characterized in osteoblast and adipocyte differentiation. KEY RESULTS: We identified 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2'-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) as a TAZ modulator. TM-25659 enhanced nuclear TAZ localization in a dose-dependent manner and attenuated PPARγ-mediated adipocyte differentiation by facilitating PPARγ suppression activity of TAZ. In addition, TAZ-induced RUNX2 activity activation was further increased in osteoblasts, causing increased osteoblast differentiation. Accordingly, TM-25659 suppressed bone loss in vivo and decreased weight gain in an obesity model. After oral administration, TM-25659 had a favourable pharmacokinetic profile. CONCLUSION AND IMPLICATIONS: TM-25659 stimulated nuclear TAZ localization and thus caused TAZ to suppress PPARγ-dependent adipogenesis and enhance RUNX2-induced osteoblast differentiation in vitro and in vivo. Our data suggest that TM-25659 could be beneficial in the control of obesity and bone loss.

Removal of dimethyl sulfide utilizing activated carbon fiber-supported photocatalyst in continuous-flow system.

The present study investigated the adsorptional photocatalytic decomposition (APD) efficiency of activated carbon fiber-supported TiO(2) (ACF/TiO(2)) in a continuous-flow reactor for the removal of dimethyl sulfide (DMS). The SEM analysis identified that the ACF/TiO(2) exhibited the same tridimensional shape as uncovered ACF and that a TiO(2) photocatalyst could be embedded in the surface of the ACF. In the absence of UV light, the time-series removal efficiencies by ACF and the ACF/TiO(2) units exhibited a similar pattern, which decreased gradually as it reached close to zero. However, the APD efficiency determined via the ACF/TiO(2) with UV light remained at nearly 60% during the remaining courses of the 13-h period, after decreasing from a maximum APD of 80%. The APD efficiencies depended upon the weights of the TiO(2) embedded into the ACFs, the UV sources, the relative humidity, and DMS input concentrations. During a long-term (219-h) APD test, the APD efficiencies dropped from 80% to ca 60% within 1h after the initiation of the APD process and then fluctuated between 52% and 60%. No byproducts were measurable or observable in the effluent gas or on the ACF/TiO(2) surface. Consequently, the continuous-flow ACF/TiO(2) system could effectively be applied to control DMS without any significant functional deterioration.

Infection of porcine cells with human herpesviruses.

Porcine organs are valuable candidate materials for xenotransplantation to humans. Long-term maintenance of well functioning transplants is a prerequisite for success. Transplanted organs may be damaged by immune reactions or by infectious agents in hosts. Human herpesviruses (HHVs) establish life-long latency in humans after a primary infection. They can be reactivated with various stimuli, including immunosuppression. This study was performed to verify the infectivity of some HHVs toward porcine cells. PK-15 cells infected with HHV-1 and HHV-2 showed cytopathology from 1 day after infection. Immunofluorescent (IF) staining of HHV-1- and HHV-2-infected PK-15 cells with respective antibodies demonstrated the expression of the respective viral antigens. Permissiveness of PK-15 to HHV-1 and -2 was confirmed by an infection test on Vero cells. Islet cells infected with HHV-5 showed no gross morphologic changes during the experimental course. A limited portion of islet cells reacted only to anti-IE1 and anti-IE2, but not to anti-UL44 or anti-gB antibody by IF staining, whereas a small portion of endothelial cells reacted to anti-IEs and anti-UL44, but not to anti-gB antibody. HHV-1 and -2 can permissively infect porcine cells, but HHV-5 infects a small proportion of cells with limited viral protein expression. HHV-4 could not transform peripheral blood mononuclear cells from miniature pigs. Collectively, because some HHVs can infect and damage porcine cells or impair their functions, HHVs should be cautiously monitored and controlled in humans when porcine cells or organs are transplanted to human beings.

Anti-inflammatory activity of p-coumaryl alcohol-gamma-O-methyl ether is mediated through modulation of interferon-gamma production in Th cells.

BACKGROUND AND PURPOSE: p-Coumaryl alcohol-gamma-O-methyl ether (CAME) was isolated from Alpinia galanga and shown to contain a phenylpropanoid structure similar to p-coumaryl diacetate (CDA). CDA is known to have antioxidant and anti-inflammatory activity, but the biochemical activities of CAME are unknown. Inflammation is mediated by inflammatory cytokine production, in particular, by CD4+ T helper cells (Th cells), but it is unclear whether phenylpropanoids affect cytokine production in Th cells. In this study, we decided to investigate the functions of CAME and CDA in CD4+ Th cells. EXPERIMENTAL APPROACH: Mouse CD4+ Th cells were isolated from C57BL6 mice and stimulated with an antibody against T cell receptors in the presence of phenylpropanoids. Cytokine production was measured by elisa and intracellular cytokine staining. Gene knockout mice and tetracycline-inducible transgenic mice were used to examine the molecular mechanisms of phenylpropanoids on modulation of cytokine production. KEY RESULTS: CAME potently reduced intracellular reactive oxygen species in Th cells, as does CDA. However, although CDA was cytotoxic, CAME selectively and potently suppresses interferon-gamma (IFNgamma) production in CD4+ Th cells, without toxicity. This effect was caused by attenuated expression of the transcription factor, T-box protein expressed in T cells (T-bet), and T-bet was essential for CAME to inhibit IFNgamma production in CD4+ Th cells. CONCLUSIONS AND IMPLICATIONS: CAME selectively and substantially suppresses IFNgamma production in CD4+ Th cells by decreasing T-bet expression. As increased IFNgamma production by CD4+ Th cells can mediate inflammatory immune responses, a selective IFNgamma suppressor, such as CAME may be an effective, naturally occurring, compound for modulating inflammatory immune disorders.

Evaluation of urinary N-acetyl cysteinyl allyl isothiocyanate as a biomarker for intake and bioactivity of Brussels sprouts.

Isothiocyanates (ITC), glucosinolate hydrolysis products from Brussels sprouts (BS) and other cruciferous vegetables, are considered to protect the body from cancer by induction of detoxification enzymes such as quinone reductase (QR). Urinary N-acetyl-cysteine (NAC) conjugates of ITC have been proposed as biomarkers of crucifer intake. Here we asked if dietary intake and induction of detoxification enzymes are dose-related to urinary NAC conjugate appearance. Male F344 rats (4/group) received an AIN 76B-40 diet containing 0, 10 or 20% freeze-dried BS for 6 days. A human subject ingested 500 g BS. Urinary AITC-NAC was identified in human and rat urine. Ten and 20% BS diets caused a 1.4- and 2.3-fold induction of QR in the pancreas, a 1.5- and 2.5-fold induction in liver and a 3.1- and 3.6-fold induction in colonic epithelium, respectively. Liver and pancreatic QR induction was dose-related, whereas induction of QR in colon was less different between the two doses. Excretion of the conjugate was dose-related only on day 1, and unrelated to dose after day 2. These results suggest that urinary NAC-AITC is a qualitative biomarker for ingestion and bioactivity of BS, but that it may not be dose-related when rats are fed continuously for 2 or more days.

Ozone and hydrogen peroxyacetic acid treatment to reduce or remove EBDCs and ETU residues in a solution.

Laboratory studies were conducted in a model system to determine the effects of ozone (1 and 3 ppm) and hydrogen peroxyacetic acid (HPA) (5 and 50 ppm) at pH 4.6, 7.0, and 10.7 and at 10 and 21 degrees C on the degradation of mancozeb in solution over a 30 min period. All samples were analyzed for residues by GLC and HPLC. Ozonation and HPA treatment were effective in degrading mancozeb in solution. Rate of mancozeb degradation was dependent on pH, with the fastest rate at pH 7.0. Ethylenethiourea (ETU) residue concentrations in the mancozeb solutions were monitored over 60 min. Under controlled conditions, the ETU residue concentrations increased during the 15 min reaction time and then decreased for all three pH values. At 3 ppm of ozone treatment, no ETU residues were detected at all three pH ranges after 15 min of reaction time. Degradation of ETU by HPA was greatest at pH 4.6, and no ETU residues remained after 5 min at either 5 or 50 ppm. The results showed that ozone and HPA gave excellent degradation of pesticide residues depending on pH and temperature. These experiments indicated the potential for the removal of pesticide residues on fruit and in processed products.

Postharvest treatments for the reduction of mancozeb in fresh apples.

The objective of this study was to determine the effectiveness of chlorine, chlorine dioxide, ozone, and hydrogen peroxyacetic acid (HPA) treatments on the degradation of mancozeb and ethylenethiourea (ETU) in apples. This study was based on model experiments at neutral pH and temperature. Fresh apples were treated with two different levels of mancozeb (1 and 10 microg/mL). Several of the treatments were effective in reducing or removing mancozeb and ETU residues on spiked apples. Mancozeb residues decreased 56-99% with chlorine and 36-87% with chlorine dioxide treatments. ETU was completely degraded by 500 ppm of calcium hypochlorite and 10 ppm of chlorine dioxide at a 1 ppm spike level. However, at a 10 ppm spike level, the effectiveness of ETU degradation was lower than observed at 1 ppm level. Mancozeb residues decreased 56-97% with ozone treatment. At 1 and 3 ppm of ozone, no ETU residue was detected at 1 ppm of spiked mancozeb after both 3 and 30 min. HPA was also effective in degrading the mancozeb residues, with 44-99% reduction depending on treatment time and HPA concentrations. ETU was completely degraded at 500 ppm of HPA after 30 min of reaction time. These treatments indicated good potential for the removal of pesticide residues on fruit and in processed products.

Genomic alterations in tubular breast carcinomas.

Tubular carcinoma of the breast is a well-differentiated variant of invasive ductal carcinoma and has been shown to have an exceptionally favorable prognosis, as manifested by a low incidence of lymph node metastases and an excellent overall survival. It is unknown whether this subtype represents an early step along the continuum of development to a more aggressive, poorly differentiated ductal cancer, or whether these cancers are destined to remain well differentiated with limited metastatic potential. We undertook an analysis of 18 pure tubular carcinomas of the breast using comparative genomic hybridization to evaluate the chromosomal changes in these tumors. An average of 3.6 chromosomal alterations of the genome were identified per case. The most frequent change involved loss of 16q (in 78% of tumors) and gain of 1q (in 50% of tumors). All but one case with 1q gain also exhibited a concomitant 16q loss. Other frequent changes involved 16p gain in 7 of 18 cases (39%) and distal 8p loss in 5 of 18 cases (28%). Comparison with known genomic alterations in a mixed group of invasive cancers shows tubular cancer to have fewer overall chromosomal changes per tumor (P <.01), higher frequency of 16q loss (P <.001), and lower frequency of 17p loss (P =.007). These results strongly suggest that tubular carcinomas are a genetically distinct group of breast cancers.

Effect of BPV1 E2-mediated inhibition of E6/E7 expression in HPV16-positive cervical carcinoma cells.

OBJECTIVE: E6 and E7 proteins of high-risk-type human papillomavirus are major etiological agents for cervical carcinomas and are continuously expressed in those cancer cells. They inhibit cell cycle control functions by inactivating p53 and Rb proteins and also immortalize cells through the induction of telomerase activity. Expression of E6 and E7 genes in HeLa, an HPV18-positive cell line, has been shown to be inhibited by the E2 protein of bovine papillomavirus (BPV1), and this resulted in the activation of the p53-mediated growth inhibitory pathway followed by an inhibition of cell proliferation. In this study, the effect of BPV1 E2-mediated inhibition of E6 and E7 expression was examined in HPV16-positive cervical carcinoma cell lines recently established from Korean patients. METHODS: BPV1 E2 was expressed in the test cells through acute infection of an SV40-BPV1 recombinant virus. Its effect on cell proliferation was assessed through MTT and DNA synthesis assays, and the status of factors involved in cell cycle control was examined through Western blotting and reverse transcription-polymerase chain reaction. RESULTS: BPV1 E2 expression caused a significant decrease in E6/E7 transcription in all three cell lines. This was accompanied by an increase in the levels of p53 protein and activity and a decrease in the expression of Cdc25A, a Cdk2-activating phosphatase. Concomitantly, E2F1 activity and cellular DNA synthesis capacity were significantly reduced. CONCLUSIONS: These results indicate that inhibition of E6/E7 gene expression in the HPV16-positive cervical carcinoma cells induces suppression in cell proliferation by activating the growth inhibitory factors, p53 and Rb, and also by downregulating the cell cycle stimulatory factor, Cdc25A.

Characteristics of DNA-binding activity of human cytomegalovirus ppUL44.

Human cytomegalovirus (HCMV)-specific monoclonal antibody, SCMVM34, recognizes the early antigen encoded by UL44 of HCMV. This antigen is confined to the nucleus of HCMV-infected cells. This study was performed to characterize the DNA-binding activity of the protein encoded by UL44 of HCMV. The nuclear and cytoskeletal fraction of HCMV-infected cells was subjected to 0.4 M NaCl extraction, DEAE-Sephacel ion exchange chromatography, DNA-cellulose chromatography and SDS-PAGE analysis with monitoring of the reactive protein using SCMVM34 monoclonal antibody. The molecular weights of the resultant proteins were found to be 34, 40 and 52 kDa. The internal peptide fragments were isolated by tryptic digestion and reverse-phase HPLC. The internal amino acid sequence analysis of the peptides from the HPLC profile revealed that the antigen recognized by SCMVM34 monoclonal antibody was ppUL44. The reactive antigen began to be eluted from 250 mM NaCl (Tris-HCl pH 7.4) in DNA cellulose. The 34 kDa protein seems to bind to DEAE more tightly than the 52 kDa protein. The surface charge of 34 kDa might be more basic. Conclusively, the antigen recognized by SCMVM34 was the protein encoded by HCMV UL44, which was localized in the nuclei after HCMV infection, and was the DNA-binding protein with the characteristic that the surface charge of the molecule was more basic, as the molecular weights of the protein were decreased.

Rapid induction of senescence in human cervical carcinoma cells.

Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

Early characteristic findings in bowleg deformities: evaluation using magnetic resonance imaging.

We used magnetic resonance imaging (MRI) to evaluate bowleg deformities in infancy. Twenty-five tibiae of 13 infants were examined and divided into two groups based on MRI findings: group A had high intensity area in the medial epiphyseal cartilage on T2-weighted images. Group B had depression of medial physis and abnormal signal in the perichondrial region in addition to the epiphyseal lesion. At the final follow-up, all cases in group A demonstrated normal lower leg alignments, whereas five cases in group B showed characteristic roentogenographic findings of Blount's disease. The improvement rate of metaphyseal-diaphyseal angle was correlated with this classification. These findings suggested that abnormal findings in physis and perichondrial region might be preliminary findings in early stage of Blount's disease. The high intensity areas in the medial epiphyseal cartilage were commonly found among the cases with bowing deformities, which suggested that there might be a common pathomechanism between physiologic bowing and infantile Blount's disease.

Current national health insurance coverage policies for breast and ovarian cancer prophylactic surgery.

BACKGROUND: The efficacy of prophylactic mastectomy and oophorectomy in reducing breast and ovarian carcinoma has recently been reported in high-risk women. Because cost has become central to medical decision-making, this study was designed to evaluate currently existing coverage policies for these procedures. METHODS: A confidential detailed cross-sectional nationwide survey of 481 medical directors from the American Association of Health Plans, Medicare, and Medicaid was conducted. RESULTS: Of the 150 respondents, 65% (n = 97) had 100,000 or more enrolled members and 35% (n = 53) had fewer than 100,000 enrolled members. Only 44% of private plans have specific policies for coverage of prophylactic mastectomy for a strong family history of breast cancer and 38% of plans for a BRCA mutation. Only 20% of total responding plans had a policy for coverage of prophylactic oophorectomy under any clinical circumstance. Governmental carriers were significantly less likely to have any policy for prophylactic surgery (range, 2%-12%) compared with nongovernmental plans (range, 24%-44%; P < .001). No significant regional differences for coverage policies were identified (P > .05). CONCLUSIONS: Significant variations currently exist for health insurance coverage of prophylactic mastectomy and oophorectomy. As genetic testing becomes widespread, more uniform policies should be established to enable appropriate high-risk candidates equal access and coverage for these procedures.

Little role of anti-gB antibodies in neutralizing activity of patient's sera with human cytomegalovirus (HCMV) infection.

Human cytomegalovirus (HCMV) gB is known to play important roles in cell surface attachment, virion penetration, spread of infection from cell to cell, and provocation of neutralizing antibody. This study was performed to determine the role of anti-HCMV gB antibody in overall neutralizing response in patients with HCMV infection and healthy control with past infection. HCMV gB was stably expressed in 293 cells. With the stable cell line expressing gB as a specific immunosorbent, anti-gB antibody was removed from the current and past HCMV-infected sera and the remaining neutralizing activity was measured by plaque assay. It was shown that 19-50% of the total virus-neutralizing activity of sera with past HCMV infections was derived from anti-gB antibody, but anti-gB antibody had little effect on the total serum virus-neutralizing activity in patients currently infected with HCMV. This result suggests that neutralizing antibody to HCMV gB may reflect disease status.

Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis.

In studying biological roles of interferon regulatory factor (IRF)-1 tumor suppressor in cervical carcinogenesis, we found that HPV E7 is functionally associated with IRF-1. Binding assays indicate a physical interaction between IRF-1 and HPV E7 in vivo and in vitro. The carboxyl-terminal transactivation domain of IRF-1 was required for the interaction. Transient co-expression of E7 significantly inhibits the IRF-1-mediated activation of IFN-beta promoter in NIH-3T3 cells. Co-transfection of E7 mutants reveals that the pRb-binding portion of E7 is necessary for the E7-mediated inactivation of IRF-1. It was next determined whether histone deacetylase (HDAC) is involved in the inactivation mechanism as recently suggested, where the carboxyl-terminal zinc finger domain of E7 associates with NURD complex containing HDAC. When trichostatin A, an inhibitor of HDAC, was treated, the repressing activity of E7 was released in a dose-dependent manner. Furthermore, the mutation of zinc finger abrogates such activity without effect on the interaction with IRF-1. These results suggest that HPV E7 interferes with the transactivation function of IRF-1 by recruiting HDAC to the promoter. The immune-promoting role of IRF-1 evokes the idea that our novel finding might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer.

Antiproliferative effects of retinoic acid/interferon in cervical carcinoma cell lines: cooperative growth suppression of IRF-1 and p53.

Retinoids and interferons have been implicated in the growth regulation of cervical cancer cells. However, the molecular mechanisms are not fully defined. To analyze detailed mechanisms, HPV-positive (HeLa, CaSki), HPV-negative (C33A, HT-3) and non-cervical Cos-1 cell lines were treated with I microM all-trans-retinoic acid (RA) and/or 10 ng/ml interferon-gamma (IFN-gamma). The growth of RA-treated HeLa cells was less effectively suppressed than that of IFN-gamma-treated ones. A combination of RA and IFN-gamma leads to an additive antiproliferative effect on the cell growth. CaSki cell growth was also inhibited by IFN-gamma but was little stimulated by RA treatment, and the IFN effect was attenuated when IFN-gamma was combined with RA. HPV-negative C33A and HT-3 cells, which are defective in p53 and Rb, and Cos- 1 cells were weakly or not responsive to all combined treatments. The molecular mechanism underlying the differential effects of RA/IFN on HeLa and C33A cells was investigated. Combined RA/IFN-gamma treatment caused a marked increase in the level of IFN regulatory factor-1 (IRF-1) in HeLa, whereas no induction of IRF-1 was observed in C33A, consistent with the findings that IFN signaling is functional in HeLa but is defective in C33A cells. The increase of p53 in HeLa cells might account for the down-regulation of HPV-18 E6 gene expression by RA/IFN-gamma. Furthermore, RA/IFN-gamma treatment resulted in the concurrent induction of p21WAF1 CDK inhibitor and dephosphorylation of Rb protein. Transient co-expression of IRF-1 and p53 led to the cooperative activation of the p21WAF1 promoter. Our results indicate that 2 transcription factors, increased in response to RA/IFN-gamma, cooperate functionally to regulate the cell cycle through the activation of a common p21WAF1 gene in HeLa cells.