S-phase Induction Research Articles

Evaluation of Humulus lupulus (hops) compound anti-proliferative properties with mechanisms in human colorectal cancer cells

Colorectal cancer (CRC) is the third most diagnosed cancer in the U.S and is highly associated with daily diet and eating patterns. A plant-based diet rich in phytochemicals is known to be protective against the initiation and progression of CRC. The hop plant, a key ingredient in beer, contains diverse bioactive compounds that possess biological benefits in tumorigenesis. Xanthohumol (XN) is the most abundant prenylated flavonoid; several derivatives including isoxanthohumol (IXN), 8-prenylnaringenin (8-PN), and tetrahydroxanthohumol (TXN) have also been identified. The current study aimed to test the effect of these compounds in suppressing proliferation and to elucidate their anti-cancer mechanisms using human adenocarcinoma CRC cells. The results indicated all four hop compounds to significantly suppress the proliferation of multiple CRC cell lines. We selected TXN and XN for further study. Flow cytometry analysis indicated both to lead to significant induction of S-phase and G2/M-phase arrest. An apoptotic assay showed increases of early and late apoptosis. Western blot data indicated significant decreases of cyclin A and CDK2, increase of poly (ADP-ribose) polymerase (PARP) cleavage, and increase of CCAAT-enhancer-binding protein homologous protein (CHOP), activating transcription factor 4 (ATF4), and inositol requiring enzyme 1α (IRE1α). In addition, dose-dependent increases were observed in intracellular reactive oxygen species (ROS), mitochondrial dysfunction, and LC3II/LC3I ratio. Taken together, our study proposes an anti-cancer mechanism of TXN and XN that involves induction of ROS release, ER stress, mitochondrial dysfunction, apoptosis, and autophagy in human CRC cells.

Characterizing the tumor selectivity of oncolytic adenovirus ICVB-1042: Analysis of S-phase induction, viral replication, and cytolytic activity in human primary normal cell types.

e14503 Background: Adenoviral-based oncolytic virus (OV) therapeutics include mutations that enable activity in tumor cells and limit toxicity in normal cells. These tumor selectivity approaches often involve mutating viral E1A function or expression; however, such modifications allow viral activity in normal cells or limit anti-tumor potency. ICVB-1042 was engineered with dual modifications in E1A and E4orf6/7, resulting in superior tumor selectivity that does not compromise lytic potency in tumor cells. This improved tumor selective virus minimizes off-tumor activity and promotes robust tumor lysis. Methods: ICVB-1042 was compared to viruses with wildtype E1A and E4orf6/7 and a clinically tested virus with an E1A mutation (ICVB-2006). Viral function and killing were assessed in primary normal human renal (HRE) and bronchial (BrE) epithelial cells in the presence of the inhibitor palbociclib to pharmacologically block S-phase entry via host cell signaling. HRE and BrE were infected with virus and evaluated for indicators of viral toxicity in normal cells. Viral replication was quantified by droplet digital polymerase chain reaction (ddPCR); cell lysis was quantified by WST-1 assay. An S-phase induction assay was developed using an mCherry-Geminin reporter that allows imaging-based quantification of viral-dependent entry to S-phase. Viral activity in cells was compared with and without the CDK4/6 inhibitor using a viral-encoded fluorescent reporter to determine the requirement for host cell proliferation on viral function. Results: ICVB-1042 demonstrated superior protection in normal cells to comparator ICVB-2006 and viruses lacking tumor selectivity modifications. Palbociclib steeply reduced viral reporter gene expression (63-88% reduction), viral replication ( > 10-fold reduction), and cytolytic activity of ICVB-1042, while non-selective viruses were unaffected by this treatment. Non-selective virus infection resulted in 3.3- to 6.1-fold higher levels of S-phase entry compared to ICVB-1042. ICVB-2006 infection resembled non-selective viruses at the level of S-phase induction (3.1- to 3.3-fold above ICVB-1042) and viral reporter gene expression was modestly inhibited by palbociclib (12-31% reduction). Conclusions: The improved tumor selectivity of ICVB-1042 results in protection of normal, non-proliferative cells from viral-mediated toxicity. The conventional approach of ICVB-2006 with a single E1A mutation leaves viral replication and cell death in normal human cells mostly intact. Together, these observations support that ICVB-1042 presents normal cell protection and reduced toxicity risks associated with OV therapy.

Abstract 1498: Melanoma antigen gene A6 and androgen receptor interaction in bladder cancer

Abstract The Melanoma Antigen Gene (MAGE) protein family is a large and highly conserved group of proteins that share a common MAGE homology domain. Interestingly, several MAGE proteins are limited in expression to reproductive tissues but are aberrantly expressed in various types of human malignancies. We have recently demonstrated overexpression of MAGE family member A6 in advance-stage bladder cancer (Cancers 13:23, 5931, 2021). However, the mechanisms involved in MAGE gene overexpression and its coregulators are currently unknown. One hypothesis is that MAGEA6 and androgen receptor (AR) signaling is involved in the etiology of bladder cancer progression. In the present study, we determined the functional relationship between MAGEA6 and AR in bladder cancer. Treatment with DHT, an AR agonist, increased AR and MAGEA6 expression in UMUC3 and J82 bladder cancer cells at the protein and transcript level. On the contrary, exposure of cells to enzalutamide, an antiandrogen, resulted in simultaneous reduction of AR and MAGEA6 more prominently in UMUC3 cells, compared to J82 harboring low MAGEA6 transcript. Dual-sgRNA CRISPR/Cas9 knockout of MAGEA6 in UMUC3 and J82 cells resulted in inhibition of cell proliferation, reduced survival, and migration potential in both cell lines. An induction of S-phase and G0/G1 phase cell cycle arrest was noted in UMUC3 and J82 cells. In silico analysis showed Arg20 and Cys557 amino acid residue of AR interacts with Asp236 and Gln106 amino acid residue of MAGEA6 through hydrogen bonds. Further confirmation of physical interaction between AR and MAGEA6, performed by immunoprecipitation of AR and probed for MAGEA6 and vice versa established interaction of AR-MAGEA6 with each other which was further confirmed by mass spectrometry analysis. In conclusion, these findings confirm the oncogenic role of MAGEA6 and its physical interaction with AR may be involved in bladder cancer progression. Citation Format: Prem Prakash Kushwaha, Shiv Verma, Sanjay Gupta. Melanoma antigen gene A6 and androgen receptor interaction in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1498.

Super-resolution microscopy reveals stochastic initiation of replication in Drosophila polytene chromosomes

Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. Here, we developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. We identified replication initiation zones where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter.

Characterization of Adenovirus 5 E1A Exon 1 Deletion Mutants in the Viral Replicative Cycle.

Human adenovirus infection is driven by Early region 1A (E1A) proteins, which are the first proteins expressed following the delivery of the viral genome to the cellular nucleus. E1A is responsible for reprogramming the infected cell to support virus replication alongside the activation of expression of all viral transcriptional units during the course of the infection. Although E1A has been extensively studied, most of these studies have focused on understanding the conserved region functions outside of a full infection. Here, we investigated the effects of small deletions in E1A exon 1 on the viral replicative cycle. Almost all deletions were found to have a negative impact on viral replication with the exception of one deletion found in the mutant dl1106, which replicated better than the wild-type E1A expressing dl309. In addition to growth, we assessed the virus mutants for genome replication, induction of the cytopathic effect, gene and protein expression, sub-cellular localization of E1A mutant proteins, induction of cellular S-phase, and activation of S-phase specific cellular genes. Importantly, our study found that virus replication is likely limited by host-specific factors, rather than specific viral aspects such as the ability to replicate genomes or express late proteins, after a certain level of these has been expressed. Furthermore, we show that mutants outside of the conserved regions have significant influence on viral fitness. Overall, our study is the first comprehensive evaluation of the dl1100 series of exon 1 E1A deletion mutants in viral fitness and provides important insights into the contribution that E1A makes to viral replication in normal human cells.

A Ternary Mixture of Common Chemicals Perturbs Benign Human Breast Epithelial Cells More Than the Same Chemicals Do Individually.

As a continuous source of hormonal stimulation, environmentally ubiquitous estrogenic chemicals, ie, xenoestrogens (XEs), are a potential risk factor for breast carcinogenesis. Given their wide distribution in the environment and the fact that bisphenol-A (BPA), methylparaben (MP), and perfluorooctanoic acid (PFOA) are uniformly detected in unselected body fluid samples, it must be assumed that humans are simultaneously exposed to these chemicals almost daily. We studied the effects of a ternary mixture of BPA, MP, and PFOA on benign breast epithelial cells at the range of concentrations observed for single chemicals in human samples. Measurements of exposure impact relevant to the breast were based on endpoints associated with “hallmarks” of cancer and “key characteristics” of carcinogens. These included modulation of total estrogen receptor (ER)α, phosphorylated ERα (pERα), total ERβ, S-phase induction, and apoptotic evasion. Data from live cell measurements were fit to a log-linear dose-response model. Concentration-dependent reduction of ERβ and apoptosis evasion was observed concurrently with the induction of ERα, pERα, and S-phase fraction, and an increased rate of cell proliferation. Beyond additive effects predicted by the sum of individual test XEs, mixture treatment demonstrated synergism for the ERβ and apoptosis suppression phenotypes (p > .001). Nonmalignant breast cells were more sensitive than commonly used breast cancer lines to XE treatment in 3 of 5 endpoints. All observations were validated with cells isolated from the normal breast tissue of 14 individuals. At relatively low concentrations, a chemical mixture has striking effects on normal cell function that are missed by evaluation of single components.

The tumor suppressor RASSF1A induces the YAP1 target gene ANKRD1 that is epigenetically inactivated in human cancers and inhibits tumor growth.

The Hippo pathway regulates organ size, growth and comprises several tumor related factors, including the oncoprotein YAP1 and the tumor suppressor RASSF1A. RASSF1A is frequently epigenetically inactivated in cancer. In our study, we analyzed the effect of RASSF1A on the function of YAP1. Expression of YAP1 resulted in the downregulation of several tumor suppressor genes and induction of S-phase. Co-expression with RASSF1A normalized the expression levels of these tumor suppressors and induced a G0-G1 arrest and apoptosis. This effect was associated with the reduction of MDM2 and the increase of p53. These data suggest that the tumor suppressor RASSF1A inhibits the oncogenic potential of YAP1. Additionally, we could show that ANKRD1 is a YAP1 target gene that is induced by RASSF1A. Further analysis revealed that ANKRD1 is epigenetically inactivated in human cancer. ANKRD1 expression induced the expression of TP53 as well as BAX and CDKN1A and reduced colony formation of cancer cells. We found that ANKRD1 interacts with p53 and is involved in the destabilization of MDM2. Additionally, our data indicate that the tumor-suppressive effect of ANKRD1 depends on the presence of p53. These results suggest that ANKRD1 is a tumor-suppressive downstream target of the Hippo pathway that is epigenetically silenced in human cancer.

Characterization and clinical validation of MCM2 and TOP2A monoclonal antibodies in the BD ProEx™ C assay: An immunoassay which detects aberrant S-phase induction in cervical tissue

BackgroundThe Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DesignMonoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. ResultsAntibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. ConclusionsBD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.

Cytotoxicity and Mechanism of Action of the Marine-Derived Fungal Metabolite Trichodermamide B and Synthetic Analogues.

The trichodermamides are modified dipeptides isolated from a wide variety of fungi, including Trichoderma virens. Previous studies reported that trichodermamide B (2) initiated cytotoxicity in HCT-116 colorectal cancer cells, while trichodermamide A (1) was devoid of activity. We recently developed an efficient total synthesis for the trichodermamides A-C (1-3). Multiple intermediates and analogues were produced, and they were evaluated for biological effects to identify additional structure-activity relationships and the possibility that a simplified analogue would retain the biological effects of 2. The antiproliferative effects of 18 compounds were evaluated, and the results show that 2 and four other compounds are active in HeLa cells, with IC50 values in the range of 1.4-21 μM. Mechanism of action studies of 2 and the other active analogues revealed different spectra of activity. At the IC85 concentration, 2 caused S-phase accumulation and cell death in HeLa cells, suggesting response to DNA double-strand breaks. The analogues did not cause S-phase accumulation or induction of DNA damage repair pathways, consistent with an alternate mode of action. The mechanistic differences are hypothesized to be due to the chlorohydrin moiety in 2, which is lacking in the analogues, which could form a DNA-reactive epoxide.

Protein-Bound Polysaccharide from Corbicula fluminea Inhibits Cell Growth in MCF-7 and MDA-MB-231 Human Breast Cancer Cells.

A novel protein-bound polysaccharide, CFPS-1, isolated from Corbicula fluminea, is composed predominantly of mannose (Man) and glucose (Glc) in a molar ratio of 3.1:12.7. The polysaccharide, with an average molecular weight of about 283 kDa, also contains 10.8% protein. Atomic force microscopy, high-performance liquid chromatography, Fourier transform infrared spectroscopy, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy analyses revealed that CFPS-1 has a backbone of 1,6-linked and 1,4,6-linked-α-D-Glc, which is terminated with a 1-linked-α-D-Man residue at the O-4 position of 1,4,6-linked-α-D-Glc, in a molar ratio of 3:1:1. Preliminary in vitro bioactivity tests revealed that CFPS-1 effectively and dose-dependently inhibits human breast cancer MCF-7 and MDA-MB-231 cell growth, with an IC50 of 243 ± 6.79 and 1142 ± 14.84 μg/mL, respectively. In MCF-7, CFPS-1 produced a significant up-regulation of p53, p21, Bax and cleaved caspase-7 and down-regulation of Cdk4, cyclin D1, Bcl-2 and caspase-7. These effects resulted in cell cycle blockade at the S-phase and apoptosis induction. In contrast, in MDA-MB-231, with limited degree of change in cell cycle distribution, CFPS-1 increases the proportion of cells in apoptotic sub-G1 phase executed by down-regulation of Bcl-2 and caspase-7 and up-regulation of Bax and cleaved caspase-7. This study extends our understanding of the anticancer mechanism of C. fluminea protein-bound polysaccharide.

Abstract C52: Targeting the obesity/leptin link to TNBC: A breast cancer disparity problem

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive cancer associated with poor prognosis and is difficult to treat. Standard therapy for TNBC includes a combination of chemotherapeutic drugs: doxorubicin (DOX), paclitaxel (TAX), and cyclophosphamide (CTX). Current epidemiological data show that obesity increases TNBC incidence, which is more relevant for African American women. Recent findings suggest that leptin (an adipokine increased in obesity) is involved in TNBC acquired drug resistance via induction of breast cancer stem cells (BCSCs). Understanding the role of obesity in TNBC is vital to the development of new and effective therapies. Chemotherapeutic drugs exhibit shortcomings that may be due to increased drug resistance, survival, proliferation, and angiogenic signal redundancy induced by leptin. Our lab has developed and tested a novel, specific inhibitor of leptin signaling, LPrA2. A pegylated derivative of LPrA2 (PEG-LPrA2) has previously shown to enhance, 68 fold, LPrA2's bioavailability in mouse breast cancer models. The objective of this study was to determine the adjuvant effects of PEG-LPrA2 on TNBC in vitro. We hypothesized that PEG-LPrA2 can serve as an adjuvant for standard TNBC chemotherapeutics. This adjuvant would allow for the reduction of chemoresistance and TNBC recurrence. It could also reduce chemotherapeutic dose and undesirable side effects associated with standard therapy for the disease. Two human TNBC cell lines were cultured: MDA-MB-231 (highly metastatic) and MDA-MB-468 (minimally invasive). Cellometer imaging technologies were used to determine S-phase progression in cells treated with PEG-LPrA2 and leptin. Flow cytometry technologies were used to determine the effects of leptin on BCSC surface markers. Additionally, in vitro toxicity of PEG-LPrA2 was tested in non-malignant mammary cells (MCF-10A) via MTT assay. Our data shows that leptin (1.2 nM, equivalent to serum levels of overweight individuals) induced S-phase progression and increased BCSC markers and the formation of TNBC tumorspheres. Remarkably, PEG-LPrA2 (1.2 nM) abrogated leptin induction of S-phase, BCSC markers, and tumorspheres. It also showed no toxicity in non-malignant cells. These results suggest leptin-signaling inhibition could be used as an adjuvant strategy for chemotherapy of TNBC. These results are most relevant for obese African American women suffering from TNBC. Citation Format: Courtney D. Dill, Adriana Harbuzariu, Tia L. Harmon, Crystal C. Lipsey, Ayobami Loye, Ruben Rene Gonzalez-Perez. Targeting the obesity/leptin link to TNBC: A breast cancer disparity problem. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C52.

Emerging differential roles of the pRb tumor suppressor in trichodysplasia spinulosa-associated polyomavirus and Merkel cell polyomavirus pathogeneses

BackgroundMerkel cell carcinoma (MCC) and trichodysplasia spinulosa (TS) are two proliferative cutaneous diseases caused by the Merkel cell polyomavirus (MCPyV) and trichodysplasia spinulosa-associated polyomavirus (TSPyV) respectively. Recently, studies have elucidated a key role of the small tumor (sT) antigen in the proliferative pathogenic mechanisms of MCPyV and likely TSPyV. While both sT antigens have demonstrated a capacity in regulating cellular pathways, it remains unknown whether MCPyV and TSPyV sT antigens contribute similarly or differentially to cell proliferation. ObjectivesThe present study aims to explore the proliferative potential of MCPyV and TSPyV sT antigens by investigating their regulatory effects on the retinoblastoma protein (pRb) tumor suppressor. Study designInducible cell lines expressing MCPyV sT or TSPyV sT were created using a lentiviral packaging system. Cellular proteins were extracted and subjected to SDS-PAGE followed by Western blot detection and densitometric analysis. ResultsExpression of TSPyV sT markedly enhanced the phosphorylation of pRb in Western blot experiments. In contrast, expression of MCPyV sT did not alter pRb phosphorylation under the same experimental conditions. Densitometric analysis revealed that TSPyV sT antigen expression nearly doubled the ratio of phosphorylated to total pRb (P<0.001, Student’s T-test), while MCPyV sT antigen expression did not cause significant change in pRb phosphorylation status. ConclusionGiven that hyperphosphorylation of pRb is associated with dysregulation of the cell cycle, S-phase induction, and increased cell proliferation, our findings support an important role of TSPyV-mediated pRb deactivation in the development of TS. The observation that the pRb tumor suppressor is inactivated by TSPyV sT but not MCPyV sT provides further insights into the distinct pathobiological mechanisms of MCC and TS.

Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

Expression of ProEx C in primary and metastatic urothelial carcinoma.

ProEx C is an antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein-2. Both these proteins are over-expressed in the cell nucleus during aberrant S-phase induction of neoplastic cells, which leads to cell proliferation. The aim of this study was to determine whether ProEx C expression can detect primary and metastatic urothelial carcinoma (UC). Thirty one fine needle aspiration cell blocks (CB) with metastatic UC were identified. Immunohistochemical staining for ProEx C and thrombomodulin was performed. Additionally, staining for Pro Ex C was also performed in tissue microarrays (TMA) of 46 cases of primary UC and carcinomas from colon (80), stomach (31), pancreas (33), liver (92), ovary (24), endometrium (25), breast (60), lung (27), kidney (32), and prostate (44), as well as melanoma (22). Nuclear staining of ProEx C and membrane staining of thrombomodulin in at least 10% tumor cells was considered a positive result. Both ProEx C and thrombomodulin have similar sensitivity for metastatic UC (84% vs. 77%, p=0.75; whereas ProEx C yielded a higher sensitivity of 93% for primary UC than thrombomodulin (72%, p=0.01). In addition to UC, ProEx C is also expressed in most of the malignant neoplasms tested in our TMA study, and has the highest sensitivity in colon and stomach carcinomas (94%). ProEx C has high sensitivity for UC. However, it is also expressed in carcinomas of colon, stomach, breast, and lung carcinomas and may not be a useful marker for workup of metastatic UC.

MCM2/TOP2A (ProExC) immunohistochemistry as a predictive marker in head and neck mucosal biopsies

Mucosal biopsies from the head and neck are often small and poorly oriented, which impedes diagnostic interpretation, especially in patients with a history of cancer, being monitored for recurrence. A cocktail of antibodies targeted against DNA topoisomerase IIA and mini-chromosome maintenance protein 2 (MCM2/TOP2A, ProExC), markers of aberrant S-phase induction, have been used with success as a diagnostic adjunct in the evaluation of squamous dysplasia of the uterine cervix. We tested the utility in head and neck biopsies to see if ProExC could be used to discriminate reactive/inflammatory from true pre-neoplasia.Sixty-four archival biopsies were selected from patients who presented to the surgeon with an indication for biopsy to “rule out” dysplasia. Histologically, all biopsies showed nonspecific atypia that was difficult to discriminate from dysplasia. Twenty-three of the patients progressed to squamous carcinoma and the rest remained benign over five years follow-up. Cases stained with ProExC by IHC methods showed a significant pattern of expression (p=0.026). The staining was greatest in patients without a history of prior head and neck cancer but was not significant. Our results show that ProExC, used in conjunction with the H&E slide, can enhance the predictive power of a mucosal biopsy in a cohort of patients.

RNA Interference Characterization of Proteins Discovered by Proteomic Analysis of Pancreatic Cancer Reveals Function in Cell Growth and Survival

There is a clear need for better therapeutics and diagnostics for pancreatic cancer. We aimed to discover plasma membrane-associated proteins overexpressed in pancreatic cancer using quantitative proteomics and apply RNA interference (RNAi) to uncover proteins associated with cancer cell survival. Cell surface glycoproteins from 5 pancreatic cancer cell lines were isolated, and differential analyses were performed using mass spectrometry and the "normoid" cell line Hs766T as the comparator. For validation, immunohistochemistry was performed on tissues from 10 independent patients and 2 normal donors. Correlation of protein and mRNA expression level was determined, and functional activity characterized using RNAi. Integrin β6, CD46, tissue factor, and a novel protein, chromosome 14 open reading frame 1, were identified as overexpressed on pancreatic cancer cell lines. Immunohistochemistry demonstrated the 4 targets were overexpressed in 20% to 70% of primary pancreatic tumor specimens. Small interfering RNA knockdown resulted in a reduction of cellular proliferation by inhibiting DNA synthesis, blocking S-phase progression or induction of apoptosis. By combining a mass spectrometry identification platform and an RNAi validation platform, we have identified a panel of cell surface glycoproteins that not only are overexpressed, but also play a functional role in pancreatic tumor cell survival.

Immunohistochemical Patterns of ProEx C in Vulvar Squamous Lesions

Two major subtypes of vulvar squamous cell carcinomas (SCC) have been described. Basaloid and warty SCC are human papillomavirus-related and associated with classic vulvar intraepithelial neoplasia (VIN). Keratinizing SCC is associated with lichen sclerosus and differentiated VIN, but not with human papillomavirus. This study was undertaken to examine the expression patterns of ProEx C in vulvar SCC and its precursors. We analyzed 22 cases with normal vulvar epidermis, 13 cases of lichen sclerosus, 14 cases of condylomas, 23 cases of high-grade classic VIN, 6 cases of differentiated VIN, 3 cases of verrucous carcinomas, 10 cases of keratinizing SCC, and 8 cases of basaloid and warty SCC. ProEx C targets minichromosome maintenance protein and topoisomerase II alpha protein which are overexpressed in the cell nucleus during aberrant S-phase induction. Marked confluent ProEx C expression is present in high-grade classic VIN with nuclear staining extending into the middle and upper layers of the epidermis. Condylomas show parabasal nuclear immunoreactivity associated with scattered ProEx C-positive nuclei in the more differentiated suprabasilar layers. Invasive SCC shows variable staining patterns. In contrast, ProEx C staining is essentially limited to the basal and parabasal layers in normal epidermis, lichen sclerosus, differentiated VIN, and verrucous carcinoma. Overall, ProEx C is a useful proliferation marker for high-grade VIN analogous to the staining patterns reported in high-grade cervical intraepithelial neoplasia.

A Pilot Evaluation of a Novel Immunohistochemical Assay for Topoisomerase II-α and Minichromosome Maintenance Protein 2 Expression (ProEx C) in Cervical Adenocarcinoma In Situ, Adenocarcinoma, and Benign Glandular Mimics

The histopathologic distinction of cervical adenocarcinoma in situ (AIS) and invasive adenocarcinoma (AC) from some benign endocervical lesions can be challenging. The ProEx C antibody reagent targets nuclear proteins (minichromosome maintenance protein 2, MCM2 and topoisomerase II-alpha, TOP2A), which are over expressed during the aberrant S-phase induction of HPV infected and neoplastic cells. In this immunohistochemical study the utility of the ProEx C reagent in distinguishing AIS and AC from a variety of non-neoplastic glandular lesions was examined. ProEx C immunohistochemical staining was performed on sections from formalin-fixed, paraffin-embedded tissue of 65 cervical tissues including 48 non-neoplastic cervices (normal [n=10], microglandular hyperplasia [n=10], tubal metaplasia [n=11], cervical endometriosis [n=7], reactive endocervix [n=10]) and 17 cervices with glandular malignancy (AIS [n=12] and AC [n=5]). Both intensity and prevalence of immunoreactivity was scored. The median and distribution of scores for both prevalence and intensity was compared for AIS versus each of the 5 benign cervical lesions using a Mann-Whitney U test. The median and distribution of prevalence of immunohistochemical staining for AIS was different from all benign mimics, but the intensity of staining for AIS did overlap with some mimics as it was not significantly different from endometriosis, microglandular hyperplasia, and reactive endocervix. ProEx C reagent has potential as an adjunctive testing tool in the histopathologic diagnosis of both AIS and AC, particularly in difficult cases with small biopsies or foci of disease.

Induction of mitotic S-phase of host and neighboring cells by Toxoplasma gondii enhances parasite invasion

The intracellular parasite Toxoplasma gondii extensively modifies its host cell so as to efficiently grow and divide. Among these cellular changes, T. gondii alters the cell cycle of host cells it has invaded. We found that T. gondii affects the cell cycle of not only the cells it directly invades, but neighboring cells as well. Both direct invasion by T. gondii and exposure to filtered medium from cultures of T. gondii-infected cells (conditioned medium) caused normally quiescent fibroblasts to enter S-phase. T. gondii has been shown to attach to and invade S-phase host cells more readily, and we found that conditioned medium increased the rate of invasion of T. gondii into new host cells. Thus it appears that T. gondii directly releases, or induces parasitized host cells to release, a factor that induces neighboring cells to enter S-phase, allowing more rapid invasion by extracellular T. gondii and providing a possible selective advantage for the parasite.

Nuclear Survivin Abrogates Multiple Cell Cycle Checkpoints and Enhances Viral Oncolysis

Survivin (BIRC5) promotes cell division and survival with roles as chromosomal passenger protein and inhibitor of apoptosis protein (IAP). It is overexpressed in many cancers and is associated with resistance to chemotherapy and radiation. Previously, we showed that expression of survivin within the nucleus of HeLa cells accelerates its degradation and blocks apoptosis inhibition without affecting localization during mitosis. Here, we have investigated the effects of survivin on cell cycle control and potential therapeutic consequences using HeLa and IGROV1 cells expressing wild-type and nuclear-targeted survivin. We show that overexpression of survivin, especially within the nucleus, increases control over G(1)-S checkpoint via increased nuclear accumulation of cyclin D and cyclin-dependent kinase 4 and subsequent pRb phosphorylation. We investigated the influence of survivin on the activity of the E1A CR2-deleted oncolytic adenovirus dl922-947, which depends critically on an aberrant G(1)-S checkpoint. Nuclear expression of survivin augments virus-induced S-phase induction and increases viral protein expression and overall viral replication. There is a consequent increase in antitumor activity both in vitro and in vivo. The increased dl922-947 activity is restricted to malignant cells and is not associated with induction of apoptosis, nor does it rely on the role of survivin as an IAP. In addition, we observe the appearance of a large >or=4N population coincident with multiple mitotic defects in dl922-947-infected cells, both of which are significantly increased by nuclear survivin. This indicates that adenoviral activity is facilitated by abrogation of multiple cell cycle checkpoints and can be enhanced by expression of survivin within the nucleus.