Yuzuru Minemoto

A new PCR-based assay amplifies the E6-E7 genes of most mucosal human papillomaviruses (HPV).

We established a new assay to detect the E6-E7 DNA of mucosal human papillomaviruses (HPV) by a PCR-based method using four pairs of degenerate LCR and E7 primers (LCR-E7 PCR). This assay amplifies the full length of E6 and the N-terminal part of E7. HPV typing was performed using restriction-fragment-length polymorphism (RFLP), and by analyzing the sequences of cloned PCR products. We compared this assay with the first generation hybrid captured assay (HCA-I) and the MY09/11-PCR method. LCR-E7 PCR was able to detect more than 34 mucosal HPV types and theoretically should detect two additional types. LCR-157 PCR and HCA-I detected HPV DNA in 70% (69/99) and 55% (54/99) of low-grade cervical intraepithelial lesions (LSIL), 89% (105/118) and 76% (90/118) of high-grade cervical intraepithelial lesions (HSIL), and 90% (56/62) and 79% (49/62) of invasive squamous cell carcinomas (SCC), respectively. LCR-E7 PCR was more sensitive than the HCA-1 test. Discordant results between the LCR-E7 and MY 11/09-PCR tests were observed in one of 185 (0.5%) normal samples, seven of 85 (8.2%) LSIL samples, seven of 82 (8.5%) HSIL samples, and four of 72 (5.6%) SCC samples. The discordant results were mostly observed in samples with a low-copy number of the HPV genome or with multiple HPV infection. The sensitivity of LCR-E7 PCR was equivalent to that of MY 11/09 ECR, and false positives were less frequent in LCR-E7 PCR. LCR-E7 PCR may be useful for determining the biological activity of detected HPV types, since this method amplifies the entire E6 gene.

Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1

Vpr, an accessory gene of HIV‐1, induces cell cycle abnormality with accumulation at G2/M phase and increased ploidy. Since abnormality of mitotic checkpoint control provides a molecular basis of genomic instability, we studied the effects of Vpr on genetic integrity using a stable clone, named MIT‐23, in which Vpr expression is controlled by the tetracycline‐responsive promoter. Treatment of MIT‐23 cells with doxycycline (DOX) induced Vpr expression with a giant multinuclear cell formation. Increased micronuclei (MIN) formation was also detected in these cells. Abolishment of Vpr expression by DOX removal induced numerous asynchronous cytokinesis in the multinuclear cells with leaving MIN in cytoplasm, suggesting that the transient Vpr expression could cause genetic unbalance. Consistent with this expectation, MIT‐23 cells, originally pseudodiploid cells, became aneuploid after repeated expression of Vpr. Experiments using deletion mutants of Vpr revealed that the domain inducing MIN formation as well as multinucleation was located in the carboxy‐terminal region of Vpr protein. These results suggest that Vpr induces genomic instability, implicating the possible role in the development of AIDS‐related malignancies.—Shimura, M., Tanaka, Y., Nakamura, S., Minemoto, Y., Yamashita, K., Hatake, K., Takaku, F., Ishizaka, Y. Micronuclei formation and aneuploidyinduced by Vpr, an accessory gene of human immunodeficiency virus type 1. FASEB J. 13, 621–637 (1999)

Pro‐apoptotic ASY/Nogo‐B protein associates with ASYIP

We have previously shown that ectopic expression of the ASY/Nogo‐B gene induced apoptosis in various cancer cell lines. Nogo‐A, a splice variant of the ASY, has been reported to have an inhibitory effect on neuronal regeneration in the central nervous system. To investigate the mechanism of ASY‐induced apoptosis or inhibition of neuronal regeneration, we cloned a cDNA for the ASY‐interacting protein from the human cDNA library using the yeast two‐hybrid method, and obtained a cDNA we designated as ASYIP. The ASYIP protein contains two hydrophobic regions and a double lysine endoplasmic reticulum (ER) retrieval motif at its C‐terminus, which was shown to be identical to RTN3, a reticulon family protein of unknown function. We showed that ASY and ASYIP proteins formed a complex also in human cells. Mutational analysis indicated that both of the hydrophobic regions of the ASYIP protein were required for the association. By immunofluorescence analysis, the ASYIP protein was shown to be co‐localized with ASY in the ER. Characterization of the ASYIP gene may be very useful in clarifying the mechanism of ASY‐induced apoptosis or Nogo‐involved inhibition of neuronal regeneration in the central nervous system. J. Cell. Physiol. 196: 312–318, 2003.

Induction of long interspersed nucleotide element-1 (L1) retrotransposition by 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct

Long interspersed nucleotide element-1 (L1) is a retroelement comprising about 17% of the human genome, of which 80–100 copies are competent as mobile elements (retrotransposition: L1-RTP). Although the genetic structures modified during L1-RTP have been clarified, little is known about the cellular signaling cascades involved. Herein we found that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of the aryl hydrocarbon receptor (AhR), induces L1-RTP. Notably, RNA-interference experiments combined with back-transfection of siRNA-resistant cDNAs revealed that the induction of L1-RTP by FICZ is dependent on AhR nuclear translocator-1 (ARNT1), a binding partner of AhR, and the activation of cAMP-responsive element-binding protein. However, our extensive analyses suggested that AhR is not required for L1-RTP. FICZ stimulated the interaction of the L1-encoded open reading frame-1 (ORF1) and ARNT1, and recruited ORF1 to chromatin in a manner dependent on the activation of mitogen-activated protein kinase. Along with our additional observations that the cellular cascades for FICZ-induced L1-RTP were different from those of L1-RTP triggered by DNA damage, we propose that the presence of the cellular machinery of ARNT1 mediates L1-RTP. A possible role of ARNT1-mediated L1-RTP in the adaptation of living organisms to environmental changes is discussed.

Bafilomycin A1 induces apoptosis in PC12 cells independently of intracellular pH

PC12 cells growth‐arrested with bafilomycin A1 died showing apoptotic chromatin condensation in the nuclei. The bafilomycin A1‐induced chromatin condensation was preceded by neurite outgrowth (NOG), required higher concentrations of bafilomycin A1 than NOG, and was suppressed by cycloheximide and aurintricarboxylic acid. NH4Cl (10 mM), another acidotropic pH perturbing agent, neither induced apoptotic chromatin condensation by itself nor suppressed that induced by bafilomycin A1, suggesting that bafilomycin A1‐induced apoptosis occurs independently of intracellular pH in PC12 cells.

Evaluation of selective tumor detection by clinical magnetic resonance imaging using antibody-conjugated superparamagnetic iron oxide

Active targeting by monoclonal antibodies (mAbs) combined with nanosize superparamagnetic iron oxide (SPIO) is a promising technology for magnetic resonance imaging (MRI) diagnosis. However, the clinical applicability of this technology has not been investigated using appropriate controls. It is important to evaluate the targeting technology using widely used clinical 1.5-Tesla MRI in addition to the high-Tesla experimental MRI. In this study, we measured mAb-conjugated dextran-coated SPIO nanoparticles (CMDM) in vivo using clinical 1.5-Tesla MRI. MRI of tumor-bearing mice was performed using a simple comparison between positive and negative tumors derived from the same genetic background in each mouse. The system provided significant tumor-targeting specificity of the target tumor. To the best of our knowledge, this is the first report on the specific detection of target tumors by mAb-conjugated SPIO using clinical 1.5-Tesla MRI. Our observations provide clues for reliable active targeting using mAb-conjugated SPIO in clinical applications.

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

Vpr-induced DNA double-strand breaks: molecular mechanism and biological relevance.

We focus on the role of Vpr in inducing DNA double-strand breaks (DSBs) in the host cell. Based on the summarized findings of Vpr-induced DSBs and the finding of Vpr in the plasma of HIV-1-positive patients, we discuss the roles of Vpr in viral infection, especially viral infection of resting macrophages. We also describe the possible involvement of Vpr in non-AIDS-defining cancers, which represent an emerging crisis in HIV-1-positive patients.

Identification of SNF2h, a Chromatin-Remodeling Factor, as a Novel Binding Protein of Vpr of Human Immunodeficiency Virus Type 1

Vpr, an accessory gene of human immunodeficiency virus type 1, encodes a virion-associated nuclear protein that plays an important role in the primary viral infection of resting macrophages. It has a variety of biological functions, including roles in a cell cycle abnormality at G2/M phase, apoptosis, nuclear transfer of preintegration complex, and DNA double-strand breaks (DSBs), some of which depend on its association with the chromatin of the host cells. Given that DSB signals are postulated to be a positive factor in the viral infection, understanding the mode of chromatin recruitment of Vpr is important. Here, we identified SNF2h, a chromatin-remodeling factor, as a novel binding partner of Vpr involved in its chromatin recruitment. When endogenous SNF2h protein was extensively downregulated by SNF2h small interfering RNA (siRNA), the amount of Vpr loaded on chromatin decreased to about 30% of the control level. Biochemical analysis using a mutant Vpr suggested that Vpr binds SNF2h via HFRIG (amino acids 71–75 depicted by single letters) and the Vpr mutant lacking this motif lost the activity to induce DSB-dependent signals. Consistently, Vpr-induced DSBs were attenuated by extensive downregulaion of endogenous SNF2h. Based on these data, we discuss the role of DSB and DSB signals in the viral infection.