The Rapid Assessment of Aggregated Wastewater Samples for Genomic Surveillance of SARS-CoV-2 on a City-Wide Scale.

Throughout the course of the ongoing SARS-CoV-2 pandemic there has been a need for approaches that enable rapid monitoring of public health using an unbiased and minimally invasive means. A major way this has been accomplished is through the regular assessment of wastewater samples by qRT-PCR to detect the prevalence of viral nucleic acid with respect to time and location. Further expansion of SARS-CoV-2 wastewater monitoring efforts to include the detection of variants of interest/concern through next-generation sequencing has enhanced the understanding of the SARS-CoV-2 outbreak. In this report, we detail the results of a collaborative effort between public health and metropolitan wastewater management authorities and the University of Louisville to monitor the SARS-CoV-2 pandemic through the monitoring of aggregate wastewater samples over a period of 28 weeks. Through the use of next-generation sequencing approaches the polymorphism signatures of Variants of Concern/Interest were evaluated to determine the likelihood of their prevalence within the community on the basis of their relative dominance within sequence datasets. Our data indicate that wastewater monitoring of water quality treatment centers and smaller neighborhood-scale catchment areas is a viable means by which the prevalence and genetic variation of SARS-CoV-2 within a metropolitan community of approximately one million individuals may be monitored, as our efforts detected the introduction and emergence of variants of concern in the city of Louisville. Importantly, these efforts confirm that regional emergence and spread of variants of interest/concern may be detected as readily in aggregate wastewater samples as compared to the individual wastewater sheds. Furthermore, the information gained from these efforts enabled targeted public health efforts including increased outreach to at-risk communities and the deployment of mobile or community-focused vaccination campaigns.

Induction of interferon response by high viral loads at early stage infection may protect against severe outcomes in COVID-19 patients.

Key elements for viral pathogenesis include viral strains, viral load, co-infection, and host responses. Several studies analyzing these factors in the function of disease severity of have been published; however, no studies have shown how all of these factors interplay within a defined cohort. To address this important question, we sought to understand how these four key components interplay in a cohort of COVID-19 patients. We determined the viral loads and gene expression using high throughput sequencing and various virological methods. We found that viral loads in the upper respiratory tract in COVID-19 patients at an early phase of infection vary widely. While the majority of nasopharyngeal (NP) samples have a viral load lower than the limit of detection of infectious viruses, there are samples with an extraordinary amount of SARS-CoV-2 RNA and a high viral titer. No specific viral factors were identified that are associated with high viral loads. Host gene expression analysis showed that viral loads were strongly correlated with cellular antiviral responses. Interestingly, however, COVID-19 patients who experience mild symptoms have a higher viral load than those with severe complications, indicating that naso-pharyngeal viral load may not be a key factor of the clinical outcomes of COVID-19. The metagenomics analysis revealed that the microflora in the upper respiratory tract of COVID-19 patients with high viral loads were dominated by SARS-CoV-2, with a high degree of dysbiosis. Finally, we found a strong inverse correlation between upregulation of interferon responses and disease severity. Overall our study suggests that a high viral load in the upper respiratory tract may not be a critical factor for severe symptoms; rather, dampened antiviral responses may be a critical factor for a severe outcome from the infection.

Age-related transcriptome changes in melanoma patients with tumor-positive sentinel lymph nodes.

Age is an important factor for determining the outcome of melanoma patients. Sentinel lymph node (SLN) status is also a strong predictor of survival for melanoma. Paradoxically, older melanoma patients have a lower incidence of SLN metastasis but a higher mortality rate when compared with their younger counterparts. The mechanisms that underlie this phenomenon remain unknown. This study uses three independent datasets of RNA samples from patients with melanoma metastatic to the SLN to identify age-related transcriptome changes in SLNs and their association with outcome. Microarray was applied to the first dataset of 97 melanoma patients. NanoString was performed in the second dataset to identify the specific immune genes and pathways that are associated with recurrence in younger versus older patients. qRT-PCR analysis was used in the third dataset of 36 samples to validate the differentially expressed genes (DEGs) from microarray and NanoString. These analyses show that FOS, NR4A, and ITGB1 genes were significantly higher in older melanoma patients with positive SLNs. IRAK3- and Wnt10b-related genes are the major pathways associated with recurrent melanoma in younger and older patients with tumor-positive SLNs, respectively. This study aims to elucidate age-related differences in SLNs in the presence of nodal metastasis.

Comparative gene expression analysis in melanocytes driven by tumor cell-derived exosomes.

Abundant with organelle-like membranous structures, the tumor microenvironment is composed of cancer cells that secrete exosomes. Studies have shown that these secreted exosomes transport RNA and active molecules to other cells to reshape the tumor microenvironment and promote tumor growth. In fact, we found that exosomes derived from melanoma cells drive pre-malignant transition in primary melanocytes. However, there is little available in the scientific literature on how exosomes modulate melanocytes in the microenvironment to optimize conditions for tumor progression and metastasis. We therefore focused this current study on identifying these conditions genetically. Through RNA sequencing, we analyzed gene expression levels of melanocytes driven by exosomes derived from melanoma and lung cancer cells compared with those without exosome controls. Significant differences were found in gene expression patterns of melanocytes driven by exosomes derived from melanoma and lung cancer cells. In the melanocytes responding to exosomes derived from melanoma cells, genes of lipopolysaccharide and regulation of leukocyte chemotaxis were predominant. In the melanocytes responding to exosomes derived from lung cancer cells, genes of DNA replication and mitotic nuclear division played an important role. These results provide further mechanistic understanding of tumor progression promoted by tumor-derived exosomes. This will also help identify potential therapeutic targets for melanoma progression.

Unique Genes in Tumor-Positive Sentinel Lymph Nodes Associated with Nonsentinel Lymph Node Metastases in Melanoma.

BACKGROUND: Current risk assessment tools to estimate the risk of nonsentinel lymph node metastases after completion lymphadenectomy for a positive sentinel lymph node (SLN) biopsy in cutaneous melanoma are based on clinical and pathologic factors. We identified a novel genetic signature that can predict non-SLN metastases in patients with cutaneous melanoma staged with a SLN biopsy. METHODS: RNA was collected for tumor-positive SLNs in patients staged by SLN biopsy for cutaneous melanoma. All patients with a tumor-positive SLN biopsy underwent completion lymphadenectomy. A 1:10 case:control series of positive and negative non-SLN patients was analyzed by microarray and quantitative RT-PCR. Candidate differentially expressed genes were validated in a 1:3 case:control separate cohort of positive and negative non-SLN patients. RESULTS: The 1:10 case:control discovery set consisted of 7 positive non-SLN cases matched to 70 negative non-SLN controls. The cases and controls were similar with regards to important clinicopathologic factors, such as gender, primary tumor site, age, ulceration, and thickness. Microarray and RT-PCR identified six potential differentially expressed genes for validation. In the 40-patient separate validation set, 10 positive non-SLN patients were matched to 30 negative non-SLN controls based on gender, ulceration, age, and thickness. Five of the six genes were differentially expressed. The five gene panel identified patients at low (7.1%) and high risk (66.7%) for non-SLN metastases. CONCLUSIONS: A novel, non-SLN gene score based on differential expressed genes in a tumor-positive SLN can identify patients at high and low risk for non-SLN metastases.

AKR1C1 as a Biomarker for Differentiating the Biological Effects of Combustible from Non-Combustible Tobacco Products.

Smoking has been established as a major risk factor for developing oral squamous cell carcinoma (OSCC), but less attention has been paid to the effects of smokeless tobacco products. Our objective is to identify potential biomarkers to distinguish the biological effects of combustible tobacco products from those of non-combustible ones using oral cell lines. Normal human gingival epithelial cells (HGEC), non-metastatic (101A) and metastatic (101B) OSCC cell lines were exposed to different tobacco product preparations (TPPs) including cigarette smoke total particulate matter (TPM), whole-smoke conditioned media (WS-CM), smokeless tobacco extract in complete artificial saliva (STE), or nicotine (NIC) alone. We performed microarray-based gene expression profiling and found 3456 probe sets from 101A, 1432 probe sets from 101B, and 2717 probe sets from HGEC to be differentially expressed. Gene Set Enrichment Analysis (GSEA) revealed xenobiotic metabolism and steroid biosynthesis were the top two pathways that were upregulated by combustible but not by non-combustible TPPs. Notably, aldo-keto reductase genes, AKR1C1 and AKR1C2, were the core genes in the top enriched pathways and were statistically upregulated more than eight-fold by combustible TPPs. Quantitative real time polymerase chain reaction (qRT-PCR) results statistically support AKR1C1 as a potential biomarker for differentiating the biological effects of combustible from non-combustible tobacco products.

Sirt1 Regulates DNA Methylation and Differentiation Potential of Embryonic Stem Cells by Antagonizing Dnmt3l.

Embryonic stem cell (ESC) abnormalities in genome methylation hamper the utility of their therapeutic derivatives; however, the underlying mechanisms are unknown. Here, we show that the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, Sirt1, selectively prevents abnormal DNA methylation of some developmental genes in murine ESCs by antagonizing Dnmt3l. Transcriptome and DNA methylome analyses demonstrated that Sirt1-null (Sirt1-/-) ESCs repress expression of a subset of imprinted and germline genes concomitant with increased DNA methylation of regulatory elements. Dnmt3l was highly expressed in Sirt1-/- ESCs, and knockdown partially rescued abnormal DNA methylation of the Sirt1 target genes. The Sirt1 protein suppressed transcription of Dnmt3l and physically interacted with the Dnmt3l protein, deacetylating and destabilizing Dnmt3l protein. Sirt1 deficiency delayed neurogenesis and spermatogenesis. These differentiation delays were significantly or partially abolished by reintroduction of Sirt1 cDNA or Dnmt3l knockdown. This study sheds light on mechanisms that restrain DNA methylation of developmentally vital genes operating in ESCs.

Sentinel Lymph Node Genes to Predict Prognosis in Node-Positive Melanoma Patients.

PURPOSE: Melanoma patients with a single microscopically-positive sentinel lymph node (SLN) are classified as stage III and are often advised to undergo expensive and substantially toxic adjuvant therapy. However, the 5-year survival rate for these patients, with or without adjuvant therapy, varies from 14 to 85 %, representing a heterogeneous biological population with a variable prognosis. We aimed to identify an SLN gene signature to aid in risk stratification of patients with tumor-positive SLNs. METHODS: Microarray experiments were performed to screen SLN genes in recurrence (N = 39) versus non-recurrence (N = 58) groups in the training dataset. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) assay was applied to confirm the expression of selected SLN genes, which were further verified using an independent validation cohort (N = 30). Area under the receiver operating characteristic curve (AUC) was calculated to evaluate prognostic accuracy of the selected SLN gene panel, and the prognostic value of our SLN gene signature was also compared with the current American Joint Committee on Cancer (AJCC) staging system. RESULTS: We identified two SLN genes (PIGR and TFAP2A) that provided high prognostic accuracy in SLN-positive melanoma patients (AUC = 0.864). These two SLN genes, along with clinicopathological features, can differentiate the high- and low-risk groups in node-positive melanoma patients in this cohort. CONCLUSION: The two SLN genes, when combined with clinicopathological features, may offer a new tool for personalized patient risk assessment.

Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties.

Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME) is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM), a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME.

Combusted but not smokeless tobacco products cause DNA damage in oral cavity cells.

The aim of this work was to investigate genomic DNA damage in human oral cavity cells after exposure to different tobacco product preparations (TPPs). The oral carcinoma cell line 101A, gingival epithelial cells HGEC, and gingival fibroblasts HGF were exposed to TPM (total particulate matter from 3R4F cigarettes), ST/CAS (2S3 smokeless tobacco extract in complete artificial saliva), and NIC (nicotine). Treatments were for 24 h using TPM at its EC-50 doses, ST/CAS and NIC at doses with equi-nicotine units, and high doses for ST/CAS and NIC. Comet assays showed that TPM, but not ST/CAS or NIC, caused substantial DNA breaks in cells; only the high ST/CAS dose caused weak DNA damage. These results were confirmed by immunofluorescence for γ-H2AX protein. These data revealed that the combusted TPP caused substantial DNA damage in all cell types, whereas the two non-combusted TPPs exerted no or only minimal DNA damage. They support epidemiologic evidence on the relative risk associated with consumption of non-combusted versus combusted tobacco products, and help to understand potential genotoxic effects of such products on oral cavity cells.

Differential cell-specific cytotoxic responses of oral cavity cells to tobacco preparations.

To examine the effects of standardized (reference) tobacco preparations on human oral cavity cells, two oral squamous cell carcinoma cell lines (101A, 101B) and normal human gingival epithelial cells (HGEC) were treated with cigarette smoke total particulate matter (TPM), smokeless tobacco extracted with complete artificial saliva (ST/CAS), or whole-smoke conditioned media (WS-CM). EC-50 values, as determined by sulforhodamine B assays, varied among the cell types and agents. When normalized to nicotine content, cytotoxicity for WS-CM and TPM was higher compared to that observed with ST/CAS. Nicotine alone had no or only minimal cytotoxicity for all cell types in the applied range. Activation of pro-apoptotic caspase-3 was examined in all cell types at their respective EC-50 doses for the three agents. TPM, but not ST/CAS or WS-CM significantly activated caspase-3 in all three cell types. Fluorescence-activated cell sorting (FACS) for expression of the early apoptosis marker Annexin V and for nuclear staining by 7-aminoactinomycin (7-AAD) revealed different extents of apoptosis versus non-apoptotic cell death for the three agents. These data characterize differential responses of normal and malignant oral cells after exposure to TPM, ST/CAS, or WS-CM. They assist in understanding differential effects of combustible versus non-combustible tobacco products, and in identifying novel biomarkers for tobacco smoke exposure and effect in the oral cavity.

Identifying mRNA, microRNA and protein profiles of melanoma exosomes.

BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma.

Global gene expression analysis of very small embryonic-like stem cells reveals that the Ezh2-dependent bivalent domain mechanism contributes to their pluripotent state.

Recently, we identified a population of Oct4(+)Sca-1(+)Lin(-)CD45(-) very small embryonic-like stem cells (VSELs) in murine and human adult tissues. VSELs can differentiate in vitro into cells from all 3 germ layers and in vivo tissue-committed stem cells. Open chromatin structure of core pluripotency transcription factors (TFs) supports the pluripotent state of VSELs. However, it has been difficult to determine how primitive VSELs maintain pluripotency, owing to their limited number in adult tissues. Here, we demonstrate by genome-wide gene-expression analysis with a small number of highly purified murine bone marrow-derived VSELs that Oct4(+) VSELs (i) express a similar, yet nonidentical, transcriptome as embryonic stem cells (ESCs), (ii) highly express cell cycle checkpoint genes, (iii) express at a low level genes involved in protein turnover and mitogenic pathways, and (iv) highly express enhancer of zeste drosophila homolog 2 (Ezh2), a polycomb group protein. Furthermore, as a result of high expression of Ezh2, VSELs, like ESCs, exhibit bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental TFs, thus preventing premature activation of the lineage-committing factors. Notably, spontaneous or RNA interference-enforced downregulation of Ezh2 during VSEL differentiation removes the bivalent domain (BD) structure, which leads to de-repression of several BD-regulated genes. Therefore, we suggest that Oct4(+) VSELs, like other pluripotent stem cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Furthermore, our global survey of VSEL gene expression signature would not only advance our understanding of biological process for their pluripotency, differentiation, and quiescence but should also help to develop better protocols for ex vivo expansion of VSELs.

Inflammatory gene expression in OVE26 diabetic kidney during the development of nephropathy.

AIM: To define renal gene expression during the development of severe albuminuria in OVE26 diabetic mice. METHODS: Kidney microarray analysis was performed at 2, 4 and 8 months. Data were validated by RT-PCR, in situ hybridization and immunohistochemistry. RESULTS: Gene expression differences between control and diabetic mice increased 10-fold from 2 to 8 months. This change was most obvious for inflammatory genes. Three inflammatory genes, complement C3, VCAM1 and CD44 were upregulated more than 4-fold. Inflammatory gene expression correlated with albuminuria and C3 and CD44 increased in tubules that accumulated albumin. VCAM1 was induced in different tubules that were neither dilated nor accumulated albumin. Six of 8 genes previously reported to be markers of human advanced diabetic nephropathy and the NF-κB_IFN_x promoter module were elevated in the oldest diabetic mice. Vitamin D inhibits diabetic renal inflammation. Vitamin D and mRNA for vitamin D synthetic enzyme CYP2B1 were elevated in kidneys of young OVE26 mice. CONCLUSIONS: OVE26 mice induce inflammatory genes consistent with advanced renal disease, associated with severe albuminuria and to a greater extent than reported in other diabetic models. They provide an excellent model of diabetic nephropathy to assess the effect of induction of inflammatory proteins.

Exosome isolation for proteomic analyses and RNA profiling.

While the existence of exosomes has been known for over three decades, they have garnered recent interest due to their potential diagnostic and therapeutic relevance. The expression and release of specific tumor-derived proteins into the peripheral circulation has served as the centerpiece of cancer screening and diagnosis. Recently, tissue-associated microRNA (miRNA) has been shown to be characteristic of tumor type and developmental origin, as well as exhibit diagnostic potential. Tumors actively release exosomes, exhibiting proteins and RNAs derived from the originating cell, into the peripheral circulation and other biologic fluids. Recently, we have demonstrated the presence of miRNAs within the RNA fraction of circulating tumor-derived exosomes. Currently, in over 75 investigations compiled in ExoCarta, over 2,300 proteins and 270 miRNAs have been linked with exosomes derived from biologic fluids. Our previous work has indicated that these circulating exosomal proteins and miRNAs can serve as surrogates for the tumor cell-associated counterparts, extending their diagnostic potential to asymptomatic individuals. In this chapter, we compare currently utilized methods for purifying exosomes for postisolation analyses. The exosomes derived from these approaches were assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, circulating exosomes isolated by ExoQuick precipitation produces exosomal RNA and protein with greater purity and quantity than chromatography, ultracentrifugation, and DynaBeads. While this precipitation approach isolates exosomes in general and does not exhibit specificity for the originating cell, the increased quantity and quality of exosomal proteins and RNA should enhance the sensitivity and accuracy of down-stream analyses, such as qRT-PCR profiling of miRNA and mass spectrometric and electrophoretic analyses of exosomal proteins.

Hypoxia-induced autophagic response is associated with aggressive phenotype and elevated incidence of metastasis in orthotopic immunocompetent murine models of head and neck squamous cell carcinomas (HNSCC).

Hypoxia confers resistance to chemoradiation therapy and promotes metastasis in head and neck squamous cell carcinomas (HNSCC). We investigated the effects of hypoxia in tumor phenotype using immunocompetent murine HNSCC models. Balb/c mice were injected intraorally with murine squamous cell carcinoma cells LY-2 and B4B8. Intratumoral hypoxia fraction was evaluated by the immunohistochemical detection of hypoxic probe pimonidazole and carbonic anhydrase IX (CAIX). Tumor cell apoptosis and autophagy in hypoxic areas of these tumors were examined immunohistochemically. Hypoxia-induced apoptotic and autophagic responses in vitro were examined by treating LY2 cells with CoCl(2). B4B8 tumors exhibited a non-aggressive phenotype characterized by its slow growth rate and the lack of metastatic spread. LY2 tumors demonstrated an aggressive phenotype characterized by rapid growth rate with regional and distant metastasis. Intratumoral hypoxia fraction in B4B8 tumors was significantly lower than in LY2 tumors. The hypoxic areas in B4B8 tumors exhibited increased apoptosis rate than that of LY2 tumors. In contrast, the hypoxic areas in LY2 tumors revealed autophagy. The induction of hypoxia in vitro elicited autophagy and not apoptosis in LY2 cells. The induction of autophagy coupled with blockage of apoptosis in hypoxic areas promotes tumor cell survival and confers aggressive phenotype in immunocompetent murine HNSCC models.

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression.

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

Cigarette smoke condensate increases cathepsin-mediated invasiveness of oral carcinoma cells.

Cigarette smoke, which contains several carcinogens known to initiate and promote tumorigenesis and metastasis, is the major cause of oral cancer. Lysosomal cathepsin proteases play important roles in tumor progression, invasion and metastasis. In the present work we investigated the effects of cigarette smoke condensate (CSC) on cathepsin (B, D and L) expression and protease-mediated invasiveness in human oral squamous cell carcinoma (OSCC) cells. Our results show that treatment of OSCC cells (686Tu and 101A) with CSC activated cathepsins B, D and L in a dose-dependent manner. Both expression and activity of these cathepsins were up-regulated in CSC-exposed versus non-exposed cells. Although cathepsin L had the lowest basal level, it had the highest induction in exposed cells compared to cathepsins B and D. Suppression of CSC-induced cathepsin B and L activities by specific chemical inhibitors decreased the invasion process, suggesting that these proteases are involved in the invasion process. Overall, our results indicate that CSC activates cathepsin B and L proteolytic activity and enhances invasiveness in OSCC cells, a response that may play a role in CSC-mediated tumor progression and metastasis dissemination.

Hypoxia inhibits TRAIL-induced tumor cell apoptosis: involvement of lysosomal cathepsins.

Tumor hypoxia interferes with the efficacy of chemotherapy, radiotherapy, and tumor necrosis factor-alpha. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a potent apoptosis inducer that limits tumor growth without damaging normal cells and tissues in vivo. We present evidence for a central role of lysosomal cathepsins in hypoxia and/or TRAIL-induced cell death in oral squamous cell carcinoma (OSCC) cells. Hypoxia or TRAIL-induced activation of cathepsins (B, D and L), caspases (-3 and -9), Bid cleavage, release of Bax and cytochrome c, and DNA fragmentation were blocked independently by zVAD-fmk, CA074Me or pepstatin A, consistent with the involvement of lysosomal cathepsin B and D in cell death. Lysosome stability and mitochondrial membrane potential were reduced in hypoxia and TRAIL-induced apoptosis. However, TRAIL treatment under hypoxic condition resulted in diminished apoptosis rates compared to treatment under normoxia. This inhibitory effect of hypoxia on TRAIL-induced apoptosis may be based on preventing Bax activation and thus protecting mitochondria stability. Our data show that TRAIL or hypoxia independently triggered activation of cathepsin B and D leading to apoptosis through Bid and Bax, and suggest that hypoxic tissue regions provide a selective environment for highly apoptosis-resistant clonal cells. Molecular therapy approaches based on cathepsin inhibitors need to address this novel tumor-preventing function of cathepsins in OSCC.

Exit from arsenite-induced mitotic arrest is p53 dependent.

BACKGROUND: Arsenic is both a human carcinogen and a chemotherapeutic agent, but the mechanism of neither arsenic-induced carcinogenesis nor tumor selective cytotoxicity is clear. Using a model cell line in which p53 expression is regulated exogenously in a tetracycline-off system (TR9-7 cells) , our laboratory has shown that arsenite disrupts mitosis and that p53-deficient cells [p53(-)], in contrast to p53-expressing cells [p53(+)], display greater sensitivity to arsenite-induced mitotic arrest and apoptosis. OBJECTIVE: Our goal was to examine the role p53 plays in protecting cells from arsenite-induced mitotic arrest. METHODS: p53(+) and p53(-) cells were synchronized in G2 phase using Hoechst 33342 and released from synchrony in the presence or absence of 5 microM sodium arsenite. RESULTS: Mitotic index analysis demonstrated that arsenite treatment delayed exit from G2 in p53(+) and p53(-) cells. Arsenite-treated p53(+) cells exited mitosis normally, whereas p53(-) cells exited mitosis with delayed kinetics. Microarray analysis performed on mRNAs of cells exposed to arsenite for 0 and 3 hr after release from G2 phase synchrony showed that arsenite induced inhibitor of DNA binding-1 (ID1) differentially in p53(+) and p53(-) cells. Immunoblotting confirmed that ID1 induction was more extensive and sustained in p53(+) cells. CONCLUSIONS: p53 promotes mitotic exit and leads to more extensive ID1 induction by arsenite. ID1 is a dominant negative inhibitor of transcription that represses cell cycle regulatory genes and is elevated in many tumors. ID1 may play a role in the survival of arsenite-treated p53(+) cells and contribute to arsenic carcinogenicity.