Nicole Benoit

Increased Mitochondrial DNA Content in Saliva Associated with Head and Neck Cancer

Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure. We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II r = 0.33, P = 0.002, respectively, Pearsons correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV‐1) to interfere with host‐cell immune effector functions. The 27‐kD Nef protein has been shown to down‐modulate specific genes of the major histocompatibility complex class I (MHC‐I) on the surface of infected pimary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV‐1. Yet, whether Nef modulates MHC‐I expression on HIV‐infected primary macrophages remains unclear. Currently available infectious HIV‐1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage‐tropic green fluorescent protein (GFP)‐tagged HIV‐1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)‐A2 on the surface of productively infected macrophages. The reporter viral genomes were replication‐competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte‐derived macrophages (MDM) after more than 2 months postinfection. Fluorescence‐activated cell sorter analyses of infected macrophages and T cells revealed that although wild‐type reporter virus infection induced a statistically significant decrease in the density of surface HLA‐A2, down‐regulation of HLA‐A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA‐A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA‐A2 down‐modulation are similar in primary T cells and macrophages.

Epstein‐Barr Virus in Head and Neck Cancer Assessed by Quantitative Polymerase Chain Reaction

Objectives/Hypothesis: Epstein‐Barr virus (EBV) has classically been associated with nasopharyngeal carcinoma and Burkitts lymphoma. Recently, multiple studies have been published linking EBV with oral squamous cell carcinoma and, to a lesser extent, hypopharyngeal and laryngeal tumors. Using a sensitive method of detection, the authors sought to analyze the presence and quantity of EBV DNA in a large cohort of head and neck cancers.

Real-Time Gap Ligase Chain Reaction: A Rapid Semiquantitative Assay for Detecting p53 Mutation at Low Levels in Surgical Margins and Lymph Nodes from Resected Lung and Head and Neck Tumors

Purpose: We have developed a real-time semiquantitative gap ligase chain reaction for detecting p53 point mutations at low level in a background of excess of wild-type DNA. Experimental Design: This method was validated by direct comparison to a previously validated but cumbersome phage plaque hybridization assay. Forty-one surgical margins and lymph nodes from 10 cases of head and neck squamous cell carcinoma and lung carcinoma were tested for p53 mutant clones. Results: Both methods detected p53 mutants in margins from 8 of the 10 cases, whereas standard pathology detected cancer cells in only 3 cases. Positive margins included tissue samples with a tumor/normal DNA ratio of up to 1:1000. Conclusions: This novel molecular approach can be performed in <5 h facilitating intraoperative use for real-time surgical resection.

Presence of 5-methylcytosine in CpNpG trinucleotides in the human genome.

While the methylation machinery of mammalian cells has been shown to be capable of both maintenance and de novo methylation at CpNpG sites, CpNpG methylation in the human genome has not been demonstrated. Here, we report the first observation of 5-methylcytosines in CpNpG triplets in the human genome. We identify the existence of CpNpG methylation in a number of genes which contain trinucleotide repeat regions, including the androgen receptor (AR). We further analyzed DNA extracted from primary tissue samples and found the same pattern of CpNpG methylation. To confirm our results, we performed Southern blot analysis by analyzing the cleavage sites of restriction enzymes within exon 1 of the AR gene and found direct evidence of the presence of 5mCs in CpNpG triplets in the human genome. Our results also suggest that this methylation pattern may be due to the human DNA methyltransferases DNMT1 and DNMT3A. Although the functional significance needs to be tested further, the discovery of inheritable CpNpG methylation in the human genome may have important implications in our understanding of gene regulation and of the development of various diseases, including cancer.

Vascular phenotyping of brain tumors using magnetic resonance microscopy (μMRI).

Abnormal vascular phenotypes have been implicated in neuropathologies ranging from Alzheimers disease to brain tumors. The development of transgenic mouse models of such diseases has created a crucial need for characterizing the murine neurovasculature. Although histologic techniques are excellent for imaging the microvasculature at submicron resolutions, they offer only limited coverage. It is also challenging to reconstruct the three-dimensional (3D) vasculature and other structures, such as white matter tracts, after tissue sectioning. Here, we describe a novel method for 3D whole-brain mapping of the murine vasculature using magnetic resonance microscopy (μMRI), and its application to a preclinical brain tumor model. The 3D vascular architecture was characterized by six morphologic parameters: vessel length, vessel radius, microvessel density, length per unit volume, fractional blood volume, and tortuosity. Region-of-interest analysis showed significant differences in the vascular phenotype between the tumor and the contralateral brain, as well as between postinoculation day 12 and day 17 tumors. These results unequivocally show the feasibility of using μMRI to characterize the vascular phenotype of brain tumors. Finally, we show that combining these vascular data with coregistered images acquired with diffusion-weighted MRI provides a new tool for investigating the relationship between angiogenesis and concomitant changes in the brain tumor microenvironment.

Colorimetric approach to high-throughput mutation analysis

High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings.

Erratum: Vascular phenotyping of brain tumors using magnetic resonance microscopy (MRI) (Journal of Cerebral Blood Flow & Metabolism (2011) 31 (1623-1636) DOI:10.1038/jcbfm.2011.17)

Results Morphologic Characterization of the Vascular Phenotype Figures 2A and 2B show the manually segmented tumor and contralateral ROIs overlaid on the MGE mMRI data, and Figures 2C and 2D show the resultant radius-encoded vascular maps for representative D12 and D17 9 L tumor-bearing brains, respectively. For tumor and contralateral ROIs of each brain, six morphologic parameters were computed as summarized in Figures 3A–3H: median vessel length, average vessel radius, MVD, LV, FV, and median vessel tortuosity. Figures 3A–3H show that with the exception of D12 LV, D17 radius and D17 tortuosity, there was a significant (P < 0.05) difference between these parameters for tumor and contralateral ROIs. For D17 brains, LV was significantly greater in tumors than in contralateral ROIs (Figure 3D); for D12 brains, radius and tortuosity were significantly greater in tumors (Figures 3B and 3F). For both D12 and D17

Abstract 5219: Relationship between intratumoral drug distribution and tumor microenvironment

Purpose: Anticancer agents released from nanocarriers have to reach cancer cells in order to demonstrate their cytotoxic activity. The distribution pattern of anticancer agents can be affected by the tumor microenvironment which varies among different types of tumors. The purpose of this study is to delineate the relationship between drug transport and tumor microenvironmental factors, such as extracellular matrix (ECM) integrity. Such studies provide insight into the factors that hinder the accessibility of cargo molecules to cancer cells. Hypothesis: In tumors with a “permissive” ECM, small molecules are likely to diffuse farther after release from nanocarriers, while they are likely to be hindered in the tumors with denser ECM. Non-invasive monitoring of drug release and subsequent intratumoral distribution of the released drug can be visualized using a dual MR contrast technique capable of distinguishing between released and intact molecules in vivo. Methods: GdDTPA was used as a surrogate tracer for a water-soluble, small molecular-weight drug. Liposomes were used as the model nanocarrier in this study. Xenografts derived from the highly-invasive and metastatic MDA-MB-231 human breast carcinoma cell line, and weakly-metastatic MCF-7 human breast carcinoma cells were used. Liposomes encapsulating GdDTPA, iron particles, and rhodamine (Lip-Gd/Fe/Rho) were prepared using a sonication method, followed by extrusion. Orthotopic xenografts were grown in female SCID mice by injecting 3×10 6 of cancer cells into the mammary fat pad of the animal. All MR studies were carried out on a horizontal bore Bruker Biospec 9.4T spectrometer. MR images were acquired before, 30 min, and 23 hrs post-i.v. administration of Lip-Gd/Fe/Rho. To identify iron particles, a 3D fast low-angle shot (FLASH) sequence was used, while a 3D fast spin echo (RARE: Rapid Acquisition with Relaxation Enhancement) sequence with an effective echo time of 50 ms and five different repetition times was acquired to track GdDTPA released from liposomes. After completion of the MRI study, tumors were excised, fixed and were cut into 10μm sections for imaging with second harmonic generation multiphoton microscopy. Region of interest analysis were carried out on the MRI data. Results/Discussion: In vivo MRI results demonstrated that a decrease in T 1 of the tumors was observed following i.v. administration of the Lip-Gd/Fe/Rho, indicating successful delivery and degradation of liposomes in the tumor. The comparison of differences in liposome delivery and its correlation with collagen-I density in the ECM of MDA-MB-231 and MCF-7 tumors is currently underway. Our results demonstrate the feasibility if using complementary imaging techniques to characterize the factors affecting intratumoral distribution of drug molecules after a release from nanocarriers. Acknowledgment: Supported by NIH grant R21 EB008162 (YK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5219.

Insufficient Tat Production Leads to Latent HIV Infection in Astrocytes

Results Following HIV infection of SVGA reporter cells, very limited infection was detected. Further, no receptor or coreceptors except CXCR4 were present on astrocytes. To see further if viral replication is blocked in astrocytes, we infected PFA or SVGA cells with VSV pseudotyped NL4-3. High levels of p24 and robust LTR-GFP or LTR-gagGFP activation were seen. VSV-HIV infected reporter cells never lost green fluorescence and green cells were negative despite of continuous low Tat and Rev production. To rule out If Tat and Rev expression were from circular unintegrated HIV-DNA, Alu PCR revealed integration of viral DNA. Further, Tat or TNF-a reactivated the latent HIV in astrocytes and the latent HIV-SVGA cells upon co-culture transmitted the infection to Jurkat cells.