The role of single nucleotide polymorphisms of the ERCC1 and MMS19 genes in predicting platinum-sensitivity, progression-free and overall survival in advanced epithelial ovarian cancer.

OBJECTIVE: This study aims to assess the role of polymorphisms in DNA repair genes, excision repair cross-complementation group 1 (ERCC1) and methyl-methanesulfonate sensitivity 19 (MMS19), in tumor response to platinum-based chemotherapy and survival in advanced epithelial ovarian cancer (EOC). METHODS: Single nucleotide polymorphism (SNP) analysis was performed on the paraffin-embedded tumor tissue of women with advanced EOC, treated with platinum-based chemotherapy at the University of Oklahoma Health Sciences Center. Polymorphisms from two ERCC1 (codon-118 and C8092A) and three MMS19 (rs2211243, rs2236575 and rs872106) gene loci were evaluated by real time PCR Allelic Discrimination Assay. RESULTS: Genotyping was performed in 107 patients, 45 platinum-sensitive and 62 platinum-resistant. ERCC1, codon-118 and C8092A genotyping was evaluable in 98 and 106 patients respectively and in all 107 patients for MMS19 polymorphisms. No differences were observed in genotype between platinum-sensitive and platinum-resistant patients. Polymorphisms in the ERCC1, codon-118 and MMS19 genes did not correlate with overall survival (OS), although a trend toward improved progression free survival (PFS) was observed in patients expressing the minor (GG) alleles of the rs872106 MMS19 gene. Women homozygous for the ERCC1-C8092A minor (AA) alleles had a significant increase in PFS compared to AC and CC patients and both AA and AC genotypes conferred improved survival over the major (CC) genotype. CONCLUSIONS: Polymorphisms in ERCC1, codon-118 and MMS19 genes are not associated with clinical response to platinum or survival. The ERCC1-C8092A genotypes containing an "A" allele were associated with significant improvement in PFS and OS strengthening the value of this specific genotype in survival.

Integration of autophagy, proteasomal degradation, unfolded protein response and apoptosis.

A single cell has the potential to kill an entire human being. Efforts to cure cancer are limited by survival of individual cancer cells despite immune surveillance and toxic therapies. Understanding the intricate network of pathways that maintain cellular homeostasis and mediate stress response or default into cell death is critical to the development of strategies to eradicate cancer. Autophagy, proteasomal degradation and the unfolded protein response (UPR) are cellular pathways that degrade and recycle excess or damaged proteins to maintain cellular homeostasis and survival. This review will discuss autophagy and how it is integrated with proteasomal degradation and UPR to govern cell fate through restoration of cellular homeostasis or default into the apoptotic cell death pathway. The first response of autophagy is macroautophagy, which sequesters cytoplasm including organelles inside double-membraned autophagosome vesicles that fuse with lysosomes to degrade and recycle the contents. Ubiquitination patterns on proteins targeted for degradation determine whether adapter proteins will bring them to developing autophagosomes or to proteasomes. Macroautophagy is followed by chaperone-mediated autophagy (CMA), in which Hsc70 (Heat shock cognate 70) selectively binds proteins with exposed KFERQ motifs and pushes them inside lysosomes through the LAMP-2A (Lysosome-associated membrane protein type 2A) receptor. These two processes and the lesser understood microautophagy, which involves direct engulfment of proteins into lysosomes, occur at basal and induced levels. Insufficient proteasome function or ER stress induction of UPR can induce autophagy, which can mitigate damage and stress. If this network is incapable of repairing the damage or overcoming continued stress, the default pathway of apoptosis is engaged to destroy the cell. Induction of macroautophagy by cancer therapeutics has led to clinical trials investigating combinations of HCQ (hydroxychloriquine) suppression of autophagy with apoptosis-inducing agents. Further study of the complex integration of autophagy, proteasomal degradation, UPR and apoptosis is likely to provide additional targets for our fight against cancer. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo.

Aberrant activation of Wingless-type (Wnt)/ß-catenin signaling is widespread in human cervical cancer. However, the underlying mechanisms of Wnt activation and the therapeutic potential of Wnt inhibition remain largely unknown. Here, we demonstrate that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, is downregulated in all human primary cervical tumors and cell lines analyzed. Our data reveal that WIF1 downregulation occurs due to promoter hypermethylation and is an early event in cervical oncogenesis. WIF1 re-expression upon 5-aza-2'-deoxycytidine treatment or WIF1 gene transfer induces significant apoptosis and G(2)/M arrest, and inhibits cervical cancer cell proliferation in vitro. Consistent with this, treatment of established mice tumor xenografts with peritumoral WIF1 gene transfer results in a significant inhibition of cancer growth and invasion. WIF1 treatment causes a significant decrease in intracellular WNT1 and TCF-4 proteins revealing novel Wnt-regulatory mechanisms. Thus, WIF1 causes a major cellular re-distribution of ß-catenin and a significant inhibition of the Wnt/ß-catenin pathway in tumor cells, as documented by a remarkable reversion in the expression of Wnt/ß-catenin transcriptional target genes (E-cadherin, c-Myc, cyclin D1, CD44 and VEGF). Consequently, multiple critical events in tumor progression and metastasis such as cell proliferation, angiogenesis and invasion were inhibited by WIF1. In addition, WIF1 modulated the expression of specific anti-apoptotic and apoptotic proteins, thereby inducing significant apoptosis in vivo. Our findings demonstrate for the first time that WIF1 downregulation by epigenetic gene silencing is an important mechanism of Wnt activation in cervical oncogenesis. Of major clinical relevance, we show that peritumoral WIF1 gene transfer reduces not only cancer growth but also invasion in well-established tumors. Therefore, our data provide novel mechanistic insights into the role of WIF1 in cervical cancer progression, and the important preclinical validation of WIF1 as a potent drug target in cervical cancer treatment.

Nonphotochemical hole-burning study of selectively stained normal and cancerous human ovarian tissues.

Results are presented of nonphotochemical hole-burning (HB) experiments on cancerous ovarian and analogous normal peritoneal in vitro tissues stained with the mitochondrial-selective dye rhodamine 800. A comparison of fluorescence excitation spectra, hole-growth kinetics data, and external electric field (Stark) effects on the shape of spectral holes burned in cancerous and normal tissues stained with rhodamine 800 revealed significant differences only in the dipole moment change (fDeltamu) measured by a combination of HB and Stark spectroscopies. It is shown that the permanent dipole moment change for the S0--> S1 transition of the rhodamine 800 molecules in cancerous tissue is higher than that of normal tissue by a factor of about 1.4. The finding is similar to the HB results obtained earlier for human ovarian surface epithelial cell lines, i.e., OV167 carcinoma and OSE(tsT)-14 normal cells stained with the same mitochondria-specific dye (Walsh et al. Biophys. J. 2003, 84, 1299). We propose that the observed difference in the permanent dipole moment change in cancerous ovarian tissue is related to a modification in mitochondrial membrane potential.

Effect of all-trans retinoic acid on tissue dynamics of choriocarcinoma cell lines: an organotypic model.

BACKGROUND: All-trans retinoic acid (ATRA) is a natural vitamin A derivative that has a profound effect on the regulation of cell growth, differentiation and death. AIM: To investigate the tissue dynamic and cellular invasion effects of ATRA in choriocarcinoma (CCA), an aggressive trophoblastic tumour, by using a three-dimensional organotypic culture model system and cell invasion assay, respectively. METHODS: An organotypic culture model of two CCA cell lines, JAR and JEG, was established. The effects of 1 microM ATRA on proliferation, differentiation and apoptosis on this CCA model were assessed by morphological assessment of the mitotic and apoptotic figures as well as by Ki-67 and caspase-related M30 cytoDeath antibody immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay. The effect of ATRA on p53 and its regulated protein product, WAF1/Cip1, was also evaluated with DO7 and p21(WAF1) antibodies, respectively. Moreover, the effect of ATRA on cellular (CCA) invasion was also investigated with Cell Invasion Kit on the JEG cell line. RESULTS: ATRA was found to induce marked apoptosis in organotypic cultures of both cell lines, as evidenced by increased M30-positive cells (p<0.0001) and increased TUNEL-positive cells (p<0.0001) in treated cultures; to decrease proliferation, as evidenced by decreased Ki-67-positive cells (p<0.0001); and to decrease p53-DO7 immunoreactivity (p<0.0001) and increase p21(WAF1) (p<0.0001) immunoreactivity. 1.5 microM ATRA was found to effectively inhibit JEG cell invasion in the cell invasion assay. CONCLUSION: ATRA treatment was found to inhibit invasion and proliferation and enhance apoptosis, probably by the activation of caspases and induction of differentiation. ATRA and synthetic retinoids may be alternative agents for the treatment of CCA.

Refining retinoids with heteroatoms.

Retinoids are a group of synthetic compounds designed to refine the numerous biological activities of retinoic acid into pharmaceuticals for several diseases, including cancer. Designs that conformationally-restricted the rotation of the structures resulted in arotinoids that were biologically active, but with increased toxicity. Incorporation of a heteroatom in one cyclic ring of the arotinoid structures drastically reduced the toxicity, while retaining biological activity. Clinical trials of a heteroarotinoid, Tazarotene, confirmed the improved chemotherapeutic ratio (efficacy/toxicity).

Heterocycle-containing retinoids. Discovery of a novel isoxazole arotinoid possessing potent apoptotic activity in multidrug and drug-induced apoptosis-resistant cells.

In a search for retinoic acid (RA) receptor ligands endowed with potent apoptotic activity, a series of novel arotinoids were prepared. Because the stereochemistry of the C9-alkenyl portion of natural 9-cis-RA and the olefinic moiety of the previously synthesized isoxazole retinoid 4 seems to have particular importance for their apoptotic activity, novel retinoid analogues with a restricted or, vice versa, a larger flexibility in this region were designed and prepared. The new compounds were evaluated in vitro for their ability to activate natural retinoid receptors and for their differentiation-inducing activity. Cytotoxic and apoptotic activities were, in addition, evaluated. In general, these analogues showed low cytotoxicity, with the restricted structures being slightly more active than the more flexible ones. As an exception, however, the isoxazole retinoid 15b proved to be particularly able to induce apoptosis at concentrations <5 microM, showing a higher activity than the classical retinoids such as all-trans-RA, 13-cis-RA, and 9-cis-RA and the previously described synthetic retinoid 4. 15b also exhibited a good affinity for the retinoid receptors. Interestingly, another important property of 15b was its ability to induce apoptosis in the HL60R multidrug-resistant (MDR) cell line, at the same concentration as is effective in HL60. Therefore, 15b represents a new retinoid possessing high apoptotic activity in an MDR cell line. The ability of 15b to act on K562 and HL60R cells suggests that this compound may have important implications in the treatment of different leukemias, and its structure could offer an interesting model for the design of new compounds endowed with apoptotic activity on MDR- and retinoid-resistant malignancies.

Effects of retinoids on cancerous phenotype and apoptosis in organotypic cultures of ovarian carcinoma.

BACKGROUND: Retinoic acid analogues, called retinoids, have shown promise in clinical trials in preventing breast and ovarian cancers. Classic retinoids bind to retinoic acid receptors, which regulate cell growth. Some novel retinoids, such as fenretinide, i.e., N-(4-hydroxyphenyl)retinamide (4-HPR), induce apoptosis through retinoic acid receptor-independent mechanisms; however, they appear to do so only at concentrations above those achieved in clinical chemoprevention trials. At lower concentrations (< or =1 microM), 4-HPR acts like classic retinoids, by inducing differentiation through a receptor-dependent mechanism. Our goal was to compare the effects of novel receptor-independent (apoptotic) retinoids with those of classic growth-inhibitory retinoids at clinically achievable doses on growth, differentiation, and apoptosis in ovarian tissue. METHODS: Four receptor-independent (apoptotic) and seven growth-inhibitory retinoids, including synthetic, low-toxicity compounds called heteroarotinoids, were administered at concentrations of 1 microM to organotypic cultures of ovarian primary and cancer cell lines: OVCAR-3, Caov-3, and SK-OV-3. After fixation, embedding, and sectioning, the growth fraction was quantified by measuring expression of the proliferation marker Ki-67/myb, differentiation was assessed by expression of mucin, and apoptosis was evaluated by the TUNEL assay. Spearman correlation analysis was performed on the data, and all P values were two-sided. RESULTS: All 11 retinoids reversed characteristics associated with the cancerous phenotype in all neoplastic cultures. Glandular structures were observed consistently in retinoid-treated, but not in untreated, OVCAR-3 and Caov-3 cultures. All retinoids decreased growth fractions, and some increased mucin expression. All receptor-independent retinoids and two receptor-dependent retinoids induced apoptosis, and the induction correlated significantly with increased expression of the mucin MUC1 (r =.83; P =.03). Retinoids with ester-linking groups did not induce apoptosis but decreased the growth fraction in correlation with MUC1 induction (r = -.93; P =.02). CONCLUSIONS: At clinically achievable concentrations, all retinoids tested decrease the growth fraction, induce differentiation and apoptosis. Induction of MUC1 expression is implicated in the mechanisms of action.

Complexity, retinoid-responsive gene networks, and bladder carcinogenesis.

Carcinogenesis involves inactivation or subversion of the normal controls of proliferation, differentiation, and apoptosis. However, these controls are robust, redundant, and interlinked at the gene expression levels, regulation of mRNA lifetimes, transcription, and recycling of proteins. One of the central systems of control of proliferation, differentiation and apoptosis is retinoid signaling. The hRAR alpha nuclear receptor occupies a central position with respect to induction of gene transcription in that when bound to appropriate retinoid ligands, its homodimers and heterodimers with hRXR alpha regulate the transcription of a number of retinoid-responsive genes. These include genes in other signaling pathways, so that the whole forms a complex network. In this study we showed that simple, cause-effect interpretations in terms of hRAR alpha gene transcription being the central regulatory event would not describe the retinoid-responsive gene network. A set of cultured bladder-derived cells representing different stages of bladder tumorigenesis formed a model system. It consisted of 2 immortalized bladder cell lines (HUC-BC and HUC-PC), one squamous cell carcinoma cell line (SCaBER), one papilloma line (RT4), and 4 transitional cell carcinomas (TCC-Sup, 5637, T24, J82) of varying stages and grades. This set of cells were used to model the range of behaviors of bladder cancers. Relative gene expression before (constitutive) and after treatment with 10 microM all-trans-retinoic acid (aTRA) was measured for androgen and estrogen receptor; a set of genes involved with retinoid metabolism and action, hRAR alpha nd beta, hRXR alpha and beta CRBP, CRABP I and II; and for signaling genes that are known to be sensitive to retinoic acid, EGFR, cytokine MK, ICAM I and transglutaminase. The phenotype for inhibition of proliferation and for apoptotic response to both aTRA and the synthetic retinoid 4-HPR was determined. Transfection with a CAT-containing plasmid containing an aTRA-sensitive promoter was used to determine if the common retinoic acid responsive element (RARE)-dependent pathway for retinoid regulation of gene expression was active. Each of the genes selected is known from previous studies to react to aTRA in a certain way, either by up- or down-regulation of the message and protein. A complex data set not readily interpretable by simple cause and effect was observed. While all cell lines expressed high levels of the mRNAs for hRXR alpha and beta that were not altered by treatment with exogenous aTRA, constitutive and stimulated responses of the other genes varied widely among the cell lines. For example, CRABP I was not expressed by J82, T24, 5637 and RT4, but was expressed at low levels that did not change in SCaBER and at moderate levels that decreased, increased, or decreased sharply in HUC-BC, TCC-Sup and HUC-PC, respectively. The expression of hRAR alpha, which governs the expression of many retinoid-sensitive genes, was expressed at moderate to high levels in all cell lines, but in some it was sharply upregulated (TCC-Sup, HUC-PC and J82), remained constant (5637 and HUC-BC), or was down-regulated (SCaBER, T24 and RT4). The phenotypes for inhibition of proliferation showed no obvious relationship to the expression of any single gene, but cell lines that were inhibited by aTRA (HUC-BC and TCC-Sup) were not sensitive to 4-HPR, and vice versa. One line (RT4) was insensitive to either retinoid. Transfection showed very little retinoid-stimulated transfection of the CAT reporter gene with RT4 or HUC-PC. About 2-fold enhancement transactivation was observed with SCaBER, HUC-BC, J82 and T24 cells and 3-8 fold with 5637, TCC-Sup cells. In HUC-BC, a G to T point mutation was found at position 606 of the hRAR alpha gene. This mutation would substitute tyrosine for asparagine in a highly conserved domain. These data indicate that retinoid signaling is probably a frequent target of inactivation in bladder carcinogenesis. (ABSTRAC

Heteroarotinoids inhibit head and neck cancer cell lines in vitro and in vivo through both RAR and RXR retinoic acid receptors.

A class of less toxic retinoids, called heteroarotinoids, was evaluated for their molecular mechanism of growth inhibition of two head and neck squamous cell carcinoma (HNSCC) cell lines SCC-2 and SCC-38. A series of 14 heteroarotinoids were screened for growth inhibition activity in vitro. The two most active compounds, one that contained an oxygen heteroatom (6) and the other a sulfur heteroatom (16), were evaluated in a xenograph model of tumor establishment in nude mice. Five days after subcutaneous injection of 10(7) SCC-38 cells, groups of 5 nu/nu mice were gavaged daily (5 days/week for 4 weeks) with 20 mg/kg/day of all-trans-retinoic acid (t-RA, 1), 10 mg/kg/day of 6, 10 mg/kg/day of 16, or sesame oil. After a few days, the dose of t-RA (1) was decreased to 10 mg/kg/day to alleviate the side effects of eczema and bone fracture. No significant toxic effects were observed in the heteroarotinoid groups. All three retinoids caused a statistically significant reduction in tumor size as determined by the Student t-test (P < 0. 05). Complete tumor regression was noted in 3 of 5 mice treated with t-RA (1), 4 of 5 mice treated with 16, 1 of 5 mice treated with 6, and 1 of 5 mice treated with sesame oil. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine that the expression levels of RARalpha, RXRalpha, and RXRbeta were similar in the two cell lines, while RARbeta expression was higher in SCC-2 over SCC-38, and RARgamma expression was higher in SCC-38 over SCC-2. Receptor cotransfection assays in CV-1 cells demonstrated that 16 was a potent activator of both RAR and RXR receptors, while 6 was selective for the RXR receptors. Transient cotransfection assays in CV-1 cells using an AP-1 responsive reporter plasmid demonstrated that t-RA (1), 6, and 16 each inhibited AP-1-driven transcription in this cell line. In conclusion, the growth inhibition activity of the RXR-selective 6 and the more potent growth inhibition activity of the RAR/RXR pan-agonist 16 implicate both RARs and RXRs in the molecular mechanism of retinoid growth inhibition. Moreover, the chemoprevention activity and the lack of toxicity of heteroarotinoids demonstrate their clinical potential in head and neck cancer chemoprevention.

Synthesis, structure-activity relationships, and RARgamma-ligand interactions of nitrogen heteroarotinoids.

Three heteroarotinoids containing a nitrogen atom in the first ring and a C-O linking group between the two aryl rings were synthesized and evaluated for RAR and RXR retinoid receptor transactivation, tumor cell growth inhibition, and transglutaminase (TGase) induction. Ethyl 4-(N,4,4-trimethyl-1,2,3,4-tetrahydroquinolinyl)benzoate (1) contained an N-CH(3) group and activated all retinoid receptors except for RARgamma. Inceasing the hydrophobicity around the rings with analogues ethyl 4-(N,4,4,7-tetramethyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (2) [7-methyl group added] and ethyl 4-(4,4-dimethyl-N-isopropyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (3) [NCH(CH(3))(2) group at C-4] increased the potency and specificity for RARalpha, RARbeta, and RXRalpha, compared to 1, but had little effect on RXRbeta and RXRgamma activation. Although 1 and 3 were unable to activate RARgamma, 2 did activate this receptor with efficacy and high potency equal to that of 9-cis-retinoic acid (9-c-RA). All three heteroarotinoids exhibited 5-8-fold greater specificities for RARbeta over RARalpha. In addition, esters 1-3 inhibited the growth of two cell lines each derived from cervix, vulvar, ovarian, and head/neck tumors with similar efficiencies to that of 9-c-RA through a mechanism independent of apoptosis. The vulvar cell lines were the most sensitive, and the ovarian lines were the least sensitive. Ester 2 was similar to 1 and 3 except that 2 was a much more potent growth inhibitor of the two vulvar cell lines, which is consistent with strong RARgamma activation by 2 (but not by 1 and 3) and the high levels of RARgamma expression in skin. All three heteroarotinoids induced production of TGase, a marker of retinoid activity in human erythroleukemic cells. Esters 2 and 3 were the more potent TGase activators than 1, in agreement with the stronger activation of the RAR receptors by 2 and 3. The biological activities of these agents, and the RARgamma potency of 2 in particular, demonstrate the promise of these compounds as pharmaceutics for cancer and skin disorders.

The mechanism of retinoic acid radiosensitization is independent of AP-1 repression in a cervical carcinoma cell line.

INTRODUCTION: Retinoic acid (RA) has been shown to radiosensitize some tumor cell lines. RA regulates gene expression through nuclear receptors that bind to retinoic acid response elements in gene promoters and that inhibit activator protein-1 (AP-1) transcription factor activity. OBJECTIVE: The aim of this study was to determine if the mechanism of radiosensitization of the CC-1 human cervical carcinoma cell line by 9-cis-RA (9cRA) involves repression of AP-1 activity. METHODS: The CCA reporter cell line was established from CC-1 by permanent transfection with the ColCAT reporter plasmid which consists of the chloramphenicol acetyltransferase (CAT) gene under the control of the AP-1-responsive collagenase gene promoter. CCA cultures were treated with various combinations of 9cRA, 60Co radiation, and an AP-1 inducer called O-tetradecanoylphorbol 13-acetate (TPA). Cultures were then evaluated in parallel for CAT expression as a measure of AP-1 activity and for clonogenic survival as a measure of radiosensitization. RESULTS: The CCA reporter line exhibited a radiation dose-responsive induction of AP-1 activity that was decreased by 5 microM 9cRA and increased by 50 ng/ml TPA. Simultaneous treatment with TPA and 9cRA prevented 9cRA repression of AP-1 and resulted in AP-1 activity above basal level. The 9cRA radiosensitized CC-1 cultures with a dose modification factor of 1.5. The survival of cultures treated simultaneously with TPA and 9cRA was statistically identical to that of cultures treated with 9cRA alone. CONCLUSION: Although TPA prevented AP-1 repression by 9cRA, it did not prevent radiosensitization in CCA cultures, therefore the mechanism of radiosensitization of CCA by 9cRA is independent of AP-1 repression.

Retinoid signaling in immortalized and carcinoma-derived human uroepithelial cells.

This paper investigates the presence and functionality of retinoid signaling pathways in human urinary bladder carcinoma and SV40-immortalized uroepithelial cell lines. Only two of eight cell lines were proliferation-inhibited by 10 microM of either all-trans or 13-cis-retinoic acid. Transactivation of the CAT gene under control of a retinoid-responsive element demonstrated functionality of the signaling pathway in both sensitive cell lines and four of six resistant cell lines. Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to-retinoic acid treatment together with numerous aberrations dysregulations. We conclude that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.

Ki-67 Expression in a Cervical Cancer Organotypic Model Correlates with Growth and EGF-R Expression.

OBJECTIVE: The study objective was to measure the effects of retinoid treatment on Ki-67 expression in a cervical carcinoma organotypic culture model and to determine whether a correlation exists between retinoid effects on Ki-67 expression and effects on growth and epidermal growth factor receptor (EGF-R) expression. METHODS: Organotypic cultures of the cervical carcinoma cell line were treated for 7 days with all-trans retinoic acid, 9-cis retinoic acid, or control solvent. Cultures were fixed and embedded in paraffin, and sections were stained with Ki-67 antibodies. Ki-67 expression was determined by light microscopy. RESULTS: Ki-67 expression was inhibited 25% in the organotypic culture treated with 9-cis retinoic acid and 32% in the culture treated with all-frans retinoic acid. Previous data demonstrated a 45% and 44% inhibition of EGF-R expression and a 49% and 63% inhibition of growth, respectively. DISCUSSION: The inhibition of Ki-67 expression by retinoids correlates with inhibition of EGF-R expression and growth as determined by a Pearson correlation (R = 0.88). Inhibition of Ki-67 and EGF-R demonstrates quantifiable effects of retinoids at both the membrane receptor and nuclear protein levels in our organotypic culture model.

Synthesis and characterization of heteroarotinoids demonstrate structure specificity relationships.

Heteroarotinoids are synthetic retinoids derived from trans-retinoic acid and the arotinoid structures and include a heteroatom in a five- or six-membered cyclic ring. This is the first systematic study of influences of the heteroatom, ring size, number of aryl groups, and terminal side chain on retinoid receptor specificity. Two new heteroarotinoids were synthesized and characterized. Although all heteroarotinoids activated RAR receptors, two dominant associations between structure and specificity were identified across all compounds. The six-membered ring conferred increased RARbeta specificity over the five-membered ring. The sulfur atom conferred greater specificity for RARgamma than the oxygen atom. RARalpha specificity was attenuated by a combination of influences from the heteroatom and aryl groups. In summary, the heteroatom and cyclic ring size exerted dominant effects, while the number of aryl rings and terminal side chain had attenuating effects on retinoid receptor specificity of heteroarotinoids.

The role of human papillomavirus in the development of pyogenic granulomas.

BACKGROUND: Pyogenic granulomas (lobular capillary hemangiomas) and condyloma acuminata share similar locations and risk factors. Human papillomavirus (HPV) types 6 and 11 are commonly associated with condyloma acuminata, but their association with pyogenic granulomas has not been evaluated. The purpose of this study was to determine whether pyogenic granulomas contain evidence of infection with condyloma-producing HPVs. METHODS: Polymerase chain reaction assays for the E6 and E7 gene sequences of HPV types 6 and 11 and another assay for the E7 region of HPV types 16, 31, 33, 35, 42, and 58 were used to evaluate deoxyribonucleic acid (DNA) extracted from archival pyogenic granuloma biopsies taken from cutaneous and oral epithelium. RESULTS: Neither cutaneous nor oral pyogenic granulomas contain amplifiable E6 or E7 sequences from any of these viruses. CONCLUSIONS: Pyogenic granulomas are not caused by HPV 6, 11, 16, 31, 33, 35, 42, or 58. This study does not exclude the possibility that other viruses may be responsible for these tumors.

Biologically active heteroarotinoids exhibiting anticancer activity and decreased toxicity.

A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were synthesized, and their biological activity was evaluated using tissue culture lines that have measurable responses to trans-retinoic acid (t-RA). Transglutaminase (TGase) was assessed in the human erythroleukemia cell line (GMO6141A) as an indicator of differentiation and apoptosis. Proliferation was evaluated in a human cervical cell line, CC-1, which exhibits dose-dependent alterations in growth rate in response to treatment with trans-retinoic acid. Activation of nuclear retinoic acid receptors was determined in a reporter cell line established from CC-1. The reporter line, called CC-B, contains a reporter gene controlled by a retinoic acid responsive element (RARE) and a thymidine kinase (tk) promoter. Treatment of the CC-B line with the heteroarotinoids resulted in a dose-responsive and retinoid-dependent regulation of reporter gene expression. The heteroarotinoids exhibited activity in all assays and correlated in a statistically significant manner between assays. RARE transactivation activity in CC-B cells correlated with induction of TGase in GMO6141A (R = 0.96) and with a decrease in the growth rate of CC-1 cells (R = -0.90). The ability of the selected heteroarotinoids to induce differentiation, inhibit proliferation, and activate nuclear receptors demonstrates the chemotherapeutic potential of these agents. In view of the biological activity cited, an in vivo toxicity study was conducted on male B6D2F1 mice with three heteroarotinoids, namely 8 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylthiochroman-6-yl)-2,4,6-heptatrienoic acid], 10 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylchroman-6-yl)-2, 4,6-heptatrienoic acid], and 13 [(E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid]. The mice were used with gavage of heteroarotinoids in corn oil [0.1, 0.2, 0.4, or 0.8 mg/kg] and with 0.01 or 0.05 mg/kg of TTNPB (5) [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]benzoic acid] as reference controls. The target organs affected in the mice by the three heteroarotinoids were those typically associated with t-RA (1) toxicity. The maximum tolerated dose (MTD) of 13 was 9.4 mg/kg/day, which was equal in toxicity to that of t-RA (1) and 1000-fold less toxic than TTNPB (5). The MTDs of 8 and 10 were 34 and 32 mg/kg/day, respectively, which is 3-fold less toxic than t-RA (1) and 3000-fold less toxic than TTNPB (5). The 3000-fold reduced toxicity, compared with only a 27% reduction biological activity of 8 and 10 with respect to that of TTNPB, observed in our assays indicates a good therapeutic ratio of these heteroarotinoids over the parent compound. The biological activity and reduced toxicity of these heteroartinoids demonstrate the potential efficacy as anticancer agents.

Biological assay for activity and molecular mechanism of retinoids in cervical tumor cells.

The composition and response of the retinoid signaling pathway in a human cell line (CC-1), representative of a low grade cervical carcinoma, were evaluated. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated expression of cytoplasmic retinol binding protein, CRBPI, cytoplasmic retinoic acid binding protein, CRABPII, and nuclear retinoic acid receptors, RAR alpha, RARgamma, RXR alpha, and RXRbeta, but not CRABPI or RARbeta. This pattern is similar to that of the ectocervix. Activation of endogenous nuclear receptors was evaluated in a reporter subline of CC-1, called CC-B, containing a reporter gene controlled by a retinoic acid responsive element (RARE) and thymidine kinase promoter. Retinoid treatment of CC-B resulted in dose-dependent increases in reporter gene expression. Retinoids inhibited growth at concentrations greater than 100 nM. 9-cis retinoic acid (1 nM) significantly stimulated growth. Immunohistochemical analysis of CC-B organotypic cultures demonstrated a high level of epidermal growth factor receptor (EGF-R) expression that was decreased by retinoids. The degree of RARE transactivation induced by retinoids significantly correlated with the degree of inhibition of growth (R = -0.96) and EGF-R expression (R = -0.92). The dose-dependent and retinoid-specific responses of CC-1 at the molecular and biological levels demonstrate the utility of this reporter cell line for evaluation of retinoid activities.

Epidermal growth factor receptor in vulvar malignancies and its relationship to metastasis and patient survival.

OBJECTIVE: To evaluate the level of epidermal growth factor receptor (EGF-R) expression in vulvar malignancies and to determine if a correlation exists between EGF-R levels and metastasis or patient survival. METHODS: All patients with a diagnosis of invasive squamous cell carcinoma of the vulva who were treated at our institution with a primary radical vulvectomy and inguinal lymph node dissection from 1983 to 1993 were eligible for the study. Sixty-one patients with available tissue blocks of benign vulvar epithelium, the primary malignant vulvar lesion, and groin node metastasis (when positive) were included in the study. Semiquantitative EGF-R expression was determined in a blinded fashion utilizing immunohistochemical staining of appropriate tissue samples. Survival was calculated utilizing Kaplan-Meier life table analysis based upon disease-free survival. RESULTS: A significant increase (P < 0.001) in mean EGF-R levels was demonstrated in the primary tumor (67%) versus benign vulvar epithelium (31%). In the 14 patients with lymph node metastasis, the mean EGF-R level in the primary tumor was 65% versus 88% in the metastatic lesion (P < 0.001). The likelihood of lymph node metastasis was elevated in those patients with a benign tissue EGF-R level > or =40% (P < 0.03) and in those patients with a primary tumor EGF-R level > or =90% (P < 0.025). Life table analysis revealed a cumulative disease-free survival of 45% for all patients. Disease-free survival in those patients with EGF-R levels > or =90% in the primary tumor was 25%, contrasting with a disease-free survival of 54% in those patients with EGF-R levels <90% (P < 0.05). CONCLUSIONS: There is a progressive increase in EGF-R expression from benign vulvar epithelium to primary malignant tissue to metastatic lesions within the same patient. Increased expression of EGF-R in the primary vulvar malignancy is significantly associated with lymph node metastasis and decreased patient survival. Increased expression of EGF-R in histologically benign vulvar epithelium has a significant association with lymph node metastasis and may predict decreased patient survival.

Differential retinoic acid radiosensitization of cervical carcinoma cell lines.

The potential of retinoic acid as a radiosensitizer was investigated using SiHa and CC-1 human uterine cervical carcinoma cell lines, representative of high- and low-grade lesions, respectively. SiHa was significantly (P < 0.05) radiosensitized, whereas CC-1 was not. Although 48 h of treatment with 5 microM 13-cis-retinoic acid prior to irradiation was sufficient to induce radiosensitization, continuation of treatment after irradiation significantly increased the effect (P < 0.05). Three hypotheses were tested to explain the different responses of the two lines. One hypothesis was that SiHa is more sensitive to retinoic acid than CC-1. Measurement of growth revealed that SiHa was more sensitive to growth inhibition by retinoic acid than CC-1. The second hypothesis was that retinoic acid increases the proportion of G1-phase cells in SiHa but not in CC-1. This was found not to be true, because a retinoic acid treatment schedule that induced radiosensitization did not alter cell cycle distribution profiles in the absence of radiation. The third hypothesis was that retinoic acid alters the cell cycle response of SiHa but not CC-1 to radiation. Postirradiation cell cycle profiles revealed that retinoic acid increased G1 delay in SiHa, whereas CC-1 exhibited no significant G1 delay. Both lines exhibited G2 delays that were unaffected by retinoic acid. In conclusion, radiosensitization of SiHa but not CC-1 may be explained by different sensitivities to retinoic acid and differences in postirradiation cell cycle responses. Radiosensitization at radiation doses used clinically was observed when retinoic acid was administered both before and after irradiation.