Human papilloma virus infection prior to coitarche.

OBJECTIVE: The aim of our study was to determine the prevalence and the natural course of anogenital human papilloma virus (HPV) infections in girls prior to coitarche attending an outpatient gynecological unit. STUDY DESIGN: Specimens were taken from the anogenital region of 114 unselected 4-15 year old girls who were referred consecutively for various gynecological problems. RESULTS: Four girls were excluded because of sexual abuse. Low-risk HPV-deoxyribonucleic acid (DNA) was detected in 4 girls (3.6%) and high-risk HPV DNA in 15 children (13.6%). Two girls testing positive for HPV DNA had clinical apparent warts. After 1 year, 2 children had persistent high-risk HPV DNA, and in 1 case we found a switch from high-risk to low-risk HPV DNA. CONCLUSION: Subclinical genital low- and high-risk HPV infections are common in girls without any history of sexual abuse or sexual activity. We found persistence of genital HPV infection in children, which could be a reservoir for HPV-associated diseases later in life.

Digalloylresveratrol, a new phenolic acid derivative induces apoptosis and cell cycle arrest in human HT-29 colon cancer cells.

Digalloylresveratrol (DIG) is a new synthetic ester of the naturally occurring polyhydroxyphenolic substances gallic acid and resveratrol which both exert anti-cancer activity in a number of tumor cell lines. The aim of the study was to identify the biochemical effects of DIG in HT-29 human colon cancer cells. DIG induced dose-dependently apoptosis after treatment for 72 h (40 microM DIG caused apoptosis in 45% of cells). DIG led to a substantial imbalance of deoxyribonucleoside triphosphates (dNTPs), the products of the enzyme ribonucleotide reductase (RR) and directly inhibited RR as it significantly reduced the incorporation of (14)C-labeled cytidine into the DNA of tumor cells. Furthermore, DIG affected the cell division and inhibited the transition from S to G2/M phase of the cell cycle. In contrast to resveratrol or gallic acid, DIG did not inhibit cyclooxygenases I and II. When HT-29 cells were simultaneously treated with DIG and 5-FU, the standard chemotherapeutic substance for colon cancer, additive growth inhibitory effects could be observed. With respect to the various biochemical and anti-proliferative effects of DIG in HT-29 cells, we regard DIG as a potential candidate for future treatment options of colon cancer and conclude that further preclinical and in vivo studies are warranted.

Antitumor effects of KITC, a new resveratrol derivative, in AsPC-1 and BxPC-3 human pancreatic carcinoma cells.

Pancreatic cancer is a very aggressive malignant disease due to lack of early diagnosis and chemotherapeutic resistance of the tumor cells. There is distinct evidence that food derived polyphenols possess chemopreventive effects in the development of several cancers including pancreatic carcinoma. Resveratrol is one of those phenolic compounds found in grape skins and other fruits with known anticancer activity. Various polymethoxylated resveratrol derivatives showed stronger antiproliferative effects than resveratrol in tumor cell lines. The aim of our study was to evaluate the cytotoxic and biochemical effects of a newly synthesized polymethoxylated resveratrol analogue, N-hydroxy-N'-(3,4,5-trimethoxphenyl)-3,4,5-trimethoxy-benzamidine (KITC) in two human pancreatic cancer cell lines. The human pancreatic cancer cell lines, AsPC-1 and BxPC-3 were used to test the potential inhibitory effect of the resveratrol derivative on cell proliferation and the underlying mechanisms of this effect. After 7 days of incubation, KITC inhibited the growth of AsPC-1 and BxPC-3 cells with IC(50) values of 9.6 and 8.7 microM, respectively. KITC (40 microM) arrested cells in the G0/G1 phase and depleted cells in the S phase of the cell cycle (-105% and -35% of control, respectively). KITC induced dose-dependent apoptosis in both pancreatic cancer cell lines and was found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of DNA synthesis. Employing growth inhibition assays, KITC acted synergistically with gemcitabine in both cell lines. In summary, we found that KITC exerted considerable antitumor activity against human pancreatic cancer cells and could be a promising candidate for further investigations to establish a new chemotherapeutic regimen.

Biochemical effects of piceatannol in human HL-60 promyelocytic leukemia cells--synergism with Ara-C.

Piceatannol (3,3',4,5'-tetrahydroxy-trans-stilbene; PCA) is a naturally occurring metabolite of resveratrol (3,4',5-trihydroxy-trans-stilbene; RV). In this study, we identified additional biochemical targets of PCA in human HL-60 promyelocytic leukemia cells. Incubation with PCA led to a significant proportion of apoptotic cells and caused an arrest in the G2-M phase of the cell cycle. PCA depleted intracellular dCTP and dGTP pools, and inhibited the incorporation of 14C-labeled cytidine into DNA. PCA significantly abolished all NTP pools, and sequential treatment with PCA and Ara-C yielded synergistic growth inhibitory effects because of remarkably increased Ara-CTP formation after PCA preincubation. Due to these promising results, PCA may support conventional chemotherapy of human malignancies and therefore, deserves further preclinical and in vivo testing.

N-hydroxy-N'-(3,4,5-trimethoxyphenyl)-3,4,5-trimethoxy-benzamidine, a novel resveratrol analog, inhibits ribonucleotide reductase in HL-60 human promyelocytic leukemia cells: synergistic antitumor activity with arabinofuranosylcytosine.

Resveratrol (3,4',5,-trihydroxystilbene, RV), an ingredient of wine, exhibits a broad spectrum of antiproliferative effects against human cancer cells. In order to develop a derivative with comparable effects, we modified the molecule by introducing additional methoxyl groups. The resulting novel RV analog, N-hydroxy-N'-(3,4,5-trimethoxyphenyl)-3,4,5-trimethoxybenzamidine (KITC), was investigated in HL-60 human promyelocytic leukemia cells. The induction of apoptosis was determined employing a specific Hoechst/propidium iodide double staining method and cell cycle distribution was evaluated by FACS. KITC's influence on the concentration of deoxyribonucleoside triphosphates, the products of ribonucleotide reductase (RR), was determined using the HPLC method. In addition, we analyzed the effects of KITC treatment on the incorporation of 14C-cytidine into the DNA of tumor cells in order to quantify the loss of RR in situ activity. To reveal a potential value of KITC for supporting conventional chemotherapy, we also examined whether a combination of KITC with arabinofuranosylcytosine (Ara-C) could yield synergistic growth inhibitory effects. KITC caused a dose-dependent induction of apoptosis, whereas no remarkable changes of the cell cycle distribution were observed. Incubation with KITC resulted in a significant depletion of intracellular dTTP and dATP pools and was also found to remarkably reduce the in situ activity of RR, the key enzyme of de novo DNA synthesis. In addition, KITC exhibited synergistic combination effects when applied sequentially with Ara-C. Due to these promising results, KITC deserves further preclinical and in vivo testing.

Gallic acid inhibits ribonucleotide reductase and cyclooxygenases in human HL-60 promyelocytic leukemia cells.

Gallic acid (GA) is a naturally occurring polyhydroxyphenolic compound and an excellent free radical scavenger. In this study, we examined its cytotoxic and biochemical effects on the human HL-60 promyelocytic leukemia cell line. GA caused a significant imbalance of deoxynucleosidetriphosphate (dNTP) pool sizes, indicating ribonucleotide reductase inhibition. Moreover, GA induced dose-dependent apoptosis in HL-60 cells (80microM GA led to the induction of apoptosis in 39% of cells) and attenuated progression from G0/G1 to the S phase of the cell cycle (60microM GA doubled the number of cells in G0/G1 phase from 22 to 44% when compared to untreated controls). We further determined IC(50) values of 3.5 and 4.4nM for the inhibition of cyclooxygenases I and II, respectively. When cells were simultaneously treated with GA and trimidox, another inhibitor of RR, highly synergistic growth inhibitory effects could be observed. Taken together, we identified novel biochemical effects of GA which could be the basis for further preclinical and in vivo studies.

Avemar, a nontoxic fermented wheat germ extract, induces apoptosis and inhibits ribonucleotide reductase in human HL-60 promyelocytic leukemia cells.

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to significantly improve the survival rate in patients suffering from various malignancies. We investigated its effects in human HL-60 promyelocytic leukemia cells. After 24, 48, and 72 h of incubation, Avemar inhibited the growth of HL-60 cells with IC50 values of 400, 190, and 160 microg/ml, respectively. Incubation with MSC caused dose-dependent induction of apoptosis in up to 85% of tumor cells. In addition, Avemar attenuated the progression from G2-M to G0-G1 phase of the cell cycle and was also found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of de novo DNA synthesis. We conclude that Avemar exerts a number of beneficial effects which could support conventional chemotherapy of human malignancies.

5-FdUrd-araC heterodinucleoside re-establishes sensitivity in 5-FdUrd- and AraC-resistant MCF-7 breast cancer cells overexpressing ErbB2.

ErbB2 overexpressing breast tumors have a poor prognosis and a high risk to develop chemoresistance to therapeutic treatment. "Chemoresistance" is a response of cells to toxic stress, and, although it is a common phenomenon, it is still poorly defined. However, a detailed understanding is required to target desensitized pathways and mechanisms for successful reactivation as part of a tailored therapy. To gain insight, which malfunctions contribute to chemoresistance, two mechanisms relevant for tissue homeostasis, the regulation of the cell cycle and of apoptosis, were investigated. Maternal MCF-7- and ErbB2-overexpressing MCF-7(erbB2) breast cancer cells were long term pretreated with 2'-deoxy-5-fluorodeoxyuridine (5-FdUrd) or 1-beta-d-arabinofuranosylcytosine (AraC) and the acquisition of drug-insensitivity was analyzed. A phosphate-conjugated heterodinucleoside consisting of one 5-FdUrd- and one AraC-moiety (5-fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine) was utilized as a tool to assess the type of acquired resistances. ErbB2-overexpression disrupted proper cell cycle regulation and furthermore facilitated the development of an apoptosis-refractory phenotype upon exposure to 5-FdUrd. Experiments with dimer 5-FdUrd-araC in ErbB2-overexpressing MCF-7(erbB2) cells, and also with nucleoside 5-FdUrd in maternal MCF-7 cells, evidenced that the phenotypes of resistance to cell cycle inhibition and to apoptosis induction were differently affected. The expression profile of cyclin D1 (but not that of p53, p21, or p27) correlated with the proliferative phenotypes and nuclear accumulation of apoptosis inducing factor (but not activation of caspase 7) with apoptotic phenotypes. Dimer 5-FdUrd-araC overrode acquired chemoresistances, whereas combined application of 5-FdUrd and AraC exhibited significantly less activity. Dimer 5-FdUrd-araC remained active in MCF-7 clones most likely by circumventing the prerequisite of first-step phosphorylation. The acquisition of chemoresistance encompassed the affection of apoptosis- and cell-cycle regulation to, respectively, different extents. Thus, drug-induced cell cycle arrest and apoptosis induction are independent of each other.

Generation of Peptide mimics of the epitope recognized by trastuzumab on the oncogenic protein Her-2/neu.

Immunizations with the oncogenic protein Her-2/neu elicit Abs exerting diverse biological effects--depending on epitope specificity, tumor growth may be inhibited or enhanced. Trastuzumab (herceptin) is a growth-inhibitory humanized monoclonal anti-Her-2/neu Ab, currently used for passive immunotherapy in the treatment of breast cancer. However, Ab therapies are expensive and have to be repeatedly administered for long periods of time. In contrast, active immunizations produce ongoing immune responses. Therefore, the study aims to generate peptide mimics of the epitope recognized by trastuzumab for vaccine formulation, ensuring the subsequent induction of tumor growth inhibitory Abs. We used the phage display technique to generate epitope mimics, mimotopes, complementing the screening Ab trastuzumab. Five candidate mimotopes were isolated from a constrained 10 mer library. These peptides were specifically recognized by trastuzumab, and showed distinctive mimicry with Her-2/neu in two experimental setups. Subsequently, immunogenicity of a selected mimotope was examined in BALB/c mice. Immunizations with a synthetic mimotope conjugated to tetanus toxoid resulted in Abs recognizing Her-2/neu in a blotted cell lysate as well as on the SK-BR-3 cell surface. Analogous to trastuzumab, the induced Abs caused internalization of the receptor from the cell surface to endosomal vesicles. These results indicate that the selected mimotopes are suitable for formulation of a breast cancer vaccine because the resulting Abs show similar biological features as trastuzumab.