Biophysical phenotype mixtures reveal advantages for tumor muscle invasion in vivo.

Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6ß1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.

Cohesive cancer invasion of the biophysical barrier of smooth muscle.

Smooth muscle is found around organs in the digestive, respiratory, and reproductive tracts. Cancers arising in the bladder, prostate, stomach, colon, and other sites progress from low-risk disease to high-risk, lethal metastatic disease characterized by tumor invasion into, within, and through the biophysical barrier of smooth muscle. We consider here the unique biophysical properties of smooth muscle and how cohesive clusters of tumor use mechanosensing cell-cell and cell-ECM (extracellular matrix) adhesion receptors to move through a structured muscle and withstand the biophysical forces to reach distant sites. Understanding integrated mechanosensing features within tumor cluster and smooth muscle and potential triggers within adjacent adipose tissue, such as the unique damage-associated molecular pattern protein (DAMP), eNAMPT (extracellular nicotinamide phosphoribosyltransferase), or visfatin, offers an opportunity to prevent the first steps of invasion and metastasis through the structured muscle.

A basal cell defect promotes budding of prostatic intraepithelial neoplasia.

Basal cells in a simple secretory epithelium adhere to the extracellular matrix (ECM), providing contextual cues for ordered repopulation of the luminal cell layer. Early high-grade prostatic intraepithelial neoplasia (HG-PIN) tissue has enlarged nuclei and nucleoli, luminal layer expansion and genomic instability. Additional HG-PIN markers include loss of α6ß4 integrin or its ligand laminin-332, and budding of tumor clusters into laminin-511-rich stroma. We modeled the invasive budding phenotype by reducing expression of α6ß4 integrin in spheroids formed from two normal human stable isogenic prostate epithelial cell lines (RWPE-1 and PrEC 11220). These normal cells continuously spun in culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells (expressing α6ß4 integrin, high-molecular-weight cytokeratin and p63, also known as TP63) and luminal cells that secrete PSA (also known as KLK3). Basal cells were optimally positioned relative to the laminin-332 layer as determined by spindle orientation. ß4-integrin-defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. This 3D model can be readily used to study mechanisms that disrupt laminin-332 continuity, for example, defects in the essential adhesion receptor (ß4 integrin), laminin-332 or abnormal luminal expansion during HG-PIN progression.

The Tumor Microenvironments of Lethal Prostate Cancer.

Localized prostate cancer (confined to the gland) generally is considered curable, with nearly a 100% 5-year-survival rate. When the tumor escapes the prostate capsule, leading to metastasis, there is a poorer prognosis and higher mortality rate, with 5-year survival dropping to less than 30%. A major research question has been to understand the transition from indolent (low risk) disease to aggressive (high risk) disease. In this chapter, we provide details of the changing tumor microenvironments during prostate cancer invasion and their role in the progression and metastasis of lethal prostate cancer. Four microenvironments covered here include the muscle stroma, perineural invasion, hypoxia, and the role of microvesicles in altering the extracellular matrix environment. The adaptability of prostate cancer to these varied microenvironments and the cues for phenotypic changes are currently understudied areas. Model systems for understanding smooth muscle invasion both in vitro and in vivo are highlighted. Invasive human needle biopsy tissue and mouse xenograft tumors both contain smooth muscle invasion. In combination, the models can be used in an iterative process to validate molecular events for smooth muscle invasion in human tissue. Understanding the complex and interacting microenvironments in the prostate holds the key to early detection of high-risk disease and preventing tumor invasion through escape from the prostate capsule.

The Cohesive Metastasis Phenotype in Human Prostate Cancer.

A critical barrier for the successful prevention and treatment of recurrent prostate cancer is detection and eradication of metastatic and therapy-resistant disease. Despite the fall in diagnoses and mortality, the reported incidence of metastatic disease has increased 72% since 2004. Prostate cancer arises in cohesive groups as intraepithelial neoplasia, migrates through muscle and leaves the gland via perineural invasion for hematogenous dissemination. Current technological advances have shown cohesive-clusters of tumor (also known as microemboli) within the circulation. Circulating tumor cell (CTC) profiles are indicative of disseminated prostate cancer, and disseminated tumor cells (DTC) are found in cohesive-clusters, a phenotypic characteristic of both radiation- and drug-resistant tumors. Recent reports in cell biology and informatics, coupled with mass spectrometry, indicate that the integrin adhesome network provides an explanation for the biophysical ability of cohesive-clusters of tumor cells to invade thorough muscle and nerve microenvironments while maintaining adhesion-dependent therapeutic resistance. Targeting cohesive-clusters takes advantage of the known ability of extracellular matrix (ECM) adhesion to promote tumor cell survival and represents an approach that has the potential to avoid the progression to drug- and radiotherapy-resistance. In the following review we will examine the evidence for development and dissemination of cohesive-clusters in metastatic prostate cancer.

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual ) of 3.25 min-1 , threefold faster than α3 integrin (1.0 min-1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min-1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min-1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6ß4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6ß1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc.

Schwann Cells Increase Prostate and Pancreatic Tumor Cell Invasion Using Laminin Binding A6 Integrin.

Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non-myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co-culture conditions were used to determine if myelinating or non-myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin-dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3-2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE-1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function-blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non-myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin-dependent tumor invasion on laminin.

Intracellular modifiers of integrin alpha 6p production in aggressive prostate and breast cancer cell lines.

Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent "inside-out" signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent "inside-out" signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype.

ERG overexpression and PTEN status predict capsular penetration in prostate carcinoma.

BACKGROUND: This study examines the combined effect of two common genetic alterations, ERG and PTEN, in prostate carcinoma progression. METHODS: Prostate tissue from 90 patients having unilateral capsular penetrating lesions, and a contra-lateral organ confined second lesion, were examined by immunohistochemistry for the expression of the TMPRSS2:ERG transformation product ERG and the loss of expression of PTEN, a powerful phosphatase inhibiting the PI3 kinase pathway. Multivariate logistic regression was carried out to analyze the data. RESULTS: After adjusting for Gleason score, the odds of having capsular penetration were 5.19 times higher (P = 0.015) for ERG+/PTEN- group as compared to the wild type (ERG-/PTEN+). CONCLUSIONS: This study presents the first evidence that ERG over expression and PTEN deletion is associated with greater risk of capsular penetration. Although further studies are needed, these results have the potential to change clinical assessment for prostate cancer.

Phase 3 clinical trial investigating the effect of selenium supplementation in men at high-risk for prostate cancer.

PURPOSE: This study was conducted to investigate the effect of Se supplementation on prostate cancer incidence in men at high risk for prostate cancer. METHODS: A Phase 3 randomized, double-blind, placebo-controlled clinical trial was conducted in 699 men at high risk for prostate cancer (prostate specific antigen (PSA) >4 ng/ml and/or suspicious digital rectal examination and/or PSA velocity >0.75 ng/ml/year), but with a negative prostate biopsy. Participants were randomized to receive daily oral placebo (N = 232), 200 µg selenium (N = 234), or 400 µg selenium (N = 233) as selenized yeast. They were followed every 6 months for up to 5 years. The time to diagnosis of prostate cancer was compared between treatment groups using the Cox proportional hazards model. RESULT: Compared to placebo, the hazard ratios [95% confidence intervals] for risk of developing prostate cancer in the selenium 200 µg/day or the selenium 400 µg/day group were 0.94 [0.52, 1.7] and 0.90 [0.48, 1.7], respectively. PSA velocity in the selenium arms was not significantly different from that observed in the placebo group (P = 0.18 and P = 0.17, respectively). CONCLUSION: Selenium supplementation appeared to have no effect on the incidence of prostate cancer in men at high risk. In conjunction with results of other studies, these data indicate that selenium supplementation may not have a role in prostate cancer chemoprevention.

The laminin binding integrin alpha6beta1 in prostate cancer perineural invasion.

Metastasizing prostate tumor cells invade along nerves innervating the encapsulated human prostate gland in a process known as perineural invasion. The extracellular matrix laminin class of proteins line the neural route and tumor cells escaping from the gland express the laminin binding integrin alpha6beta1 as a prominent cell surface receptor. Integrin alpha6beta1 promotes aggressive disease and supports prostate tumor cell metastasis to bone. Laminins and their integrin receptors are necessary for the development and maintenance of the peripheral nervous system, indicating the potential role for integrin receptors in directing prostate tumor cell invasion on nerves during perineural invasion.

Effect of aspirin, other NSAIDs, and statins on PSA and PSA velocity.

BACKGROUND: Aspirin, other non-steroidal anti-inflammatory drugs (NSAIDs), and statins have been associated with lower risk of prostate cancer and its progression, though results have been inconsistent. METHODS: Data from 140 men with prostate cancer enrolled in a Phase 2 clinical trial of selenium to prevent prostate cancer progression were analyzed to determine association between aspirin, other NSAIDs, or statin use with baseline serum prostate-specific antigen (PSA) levels and PSA velocity (rate of PSA change over time) using repeated measures over an average follow-up time of 3.2 years. Multiple linear regression and mixed effects models were used to model the association of medication use with PSA at baseline and with PSA velocity, respectively. RESULTS: Baseline PSA levels were significantly lower in aspirin users compared to non-users (5.17 ng/ml vs. 7.58 ng/ml, P = 0.001). This association was statistically significant in never smokers (aspirin users vs. non-users: 4.19 ng/ml vs. 8.24 ng/ml, P = 0.004) but not in ever smokers (aspirin users vs. non-users: 5.52 ng/ml vs. 7.3 ng/ml, P = 0.101). Statin and other NSAID use was not associated with baseline PSA. Aspirin, statin, or other NSAID use at baseline demonstrated a non-significant negative association with PSA velocity. CONCLUSION: These findings support an effect of aspirin use on PSA, particularly among never smokers. However, they do not suggest a protective effect on the disease and support previous findings that aspirin use may mask accurate measurement of PSA warranting consideration of washout procedures prior to testing.

Centrosome loss results in an unstable genome and malignant prostate tumors.

Localized, nonindolent prostate cancer (PCa) is characterized by large-scale genomic rearrangements, aneuploidy, chromothripsis, and other forms of chromosomal instability (CIN), yet how this occurs remains unclear. A well-established mechanism of CIN is the overproduction of centrosomes, which promotes tumorigenesis in various mouse models. Therefore, we developed a single-cell assay for quantifying centrosomes in human prostate tissue. Surprisingly, centrosome loss-which has not been described in human cancer-was associated with PCa progression. By chemically or genetically inducing centrosome loss in nontumorigenic prostate epithelial cells, mitotic errors ensued, producing aneuploid, and multinucleated cells. Strikingly, transient or chronic centrosome loss transformed prostate epithelial cells, which produced highly proliferative and poorly differentiated malignant tumors in mice. Our findings suggest that centrosome loss could create a cellular crisis with oncogenic potential in prostate epithelial cells.

A method of quantifying centrosomes at the single-cell level in human normal and cancer tissue.

Centrosome abnormalities are emerging hallmarks of cancer. The overproduction of centrosomes (known as centrosome amplification) has been reported in a variety of cancers and is currently being explored as a promising target for therapy. However, to understand different types of centrosome abnormalities and their impact on centrosome function during tumor progression, as well as to identify tumor subtypes that would respond to the targeting of a centrosome abnormality, a reliable method for accurately quantifying centrosomes in human tissue samples is needed. Here, we established a method of quantifying centrosomes at a single-cell level in different types of human tissue samples. We tested multiple anti-centriole and pericentriolar-material antibodies to identify bona fide centrosomes and multiplexed these with cell border markers to identify individual cells within the tissue. High-resolution microscopy was used to generate multiple Z-section images, allowing us to acquire whole cell volumes in which to scan for centrosomes. The normal cells within the tissue serve as internal positive controls. Our method provides a simple, accurate way to distinguish alterations in centrosome numbers at the level of single cells.

A Method to Reuse Archived H&E Stained Histology Slides for a Multiplex Protein Biomarker Analysis.

Archived Hematoxylin and Eosin (H&E) stained pathology slides are routinely stored to index formalin-fixed paraffin-embedded (FFPE) sample tissue blocks. FFPE blocks are clinically annotated human tumor specimens that can be valuable in studies decades after the tissue is collected. If stored properly, they have the potential to yield a valuable number of serial sectioned slides for diagnostic or research purposes. However, some retrospective studies are limited in scope because the tissue samples have been depleted or not enough material is available in stored blocks for serial sections. The goal of these studies was to determine if archived H&E-stained slides can be directly reutilized by optimizing methods to de-stain and then re-stain the H&E stained slides to allow the detection of several biomarkers of interest using a conjugated antibody with chromogen multiplex immunohistochemistry procedure. This simple but innovative procedure, combined with image analysis techniques, demonstrates the ability to perform precise detection of relevant markers correlated to disease progression in initially identified tumor regions in tissue. This may add clinical value in retaining H&E slides for further use.

Kallikrein 6 protease advances colon tumorigenesis via induction of the high mobility group A2 protein.

Kallikrein-related peptidase 6 (KLK6) overexpression is commonly observed in primary tumors of colorectal cancer (CRC) patients and has been associated with tumor aggressiveness, metastasis, and poor prognosis. We previously established a unique contribution of KLK6 in colon cancer metastasis via a specific network of microRNAs and mRNAs. Here we evaluated the cellular functions of KLK6 protease in Caco-2 colon adenocarcinoma cell line after introduction of the enzymatically active or inactive form of the enzyme. We found that proteolytically active KLK6 increased Caco-2 cells invasiveness in vitro and decreased the animal survival in the orthotopic colon cancer model. The active KLK6 induced phosphorylation of SMAD 2/3 proteins leading to the altered expression of the epithelial-mesenchymal transition (EMT) markers. KLK6 overexpression also induced the RNA-binding protein LIN28B and high-mobility group AT-hook 2 (HMGA2) transcription factor, two essential regulators of cell invasion and metastasis. In the CRC patients, KLK6 protein levels were elevated in the non-cancerous distant and adjacent tissues, compared to their paired tumor tissues (p < 0.0001 and p = 0.0157, respectively). Patients with mutant K-RAS tumors had significantly higher level of KLK6 protein in the luminal surface of non-cancerous distant tissue, compared to the corresponding tissues of the patients with K-RAS wild type tumors (p ≤ 0.05). Furthermore, KLK6 and HMGA2 immunohistochemistry (IHC) scores in patients' tumors and paired adjacent tissues positively correlated (Spearman correlation P < 0.01 and p = 0.03, respectively). These findings demonstrate the critical function of the KLK6 enzyme in colon cancer progression and its contribution to the signaling network in colon cancer.

Integrin α6ß4E variant is associated with actin and CD9 structures and modifies the biophysical properties of cell-cell and cell-extracellular matrix interactions.

Integrin α6ß4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-extracellular matrix (ECM) adhesion and induced migration, and coordinated by regions of the ß4C cytoplasmic domain. ß4E, a unique splice variant of ß4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique sequence of 114 amino acids instead of ß4C's canonical 1089 amino acids. We determined the distribution of α6ß4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6ß4C expressed in all basal cells, as expected, while α6ß4E expressed within a subset of luminal cells. α6ß4E expression was induced by three-dimensional culture conditions, activated Src, was reversible, and was stabilized by bortezomib, a proteasome inhibitor. α6ß4C expressed in all cells during induced migration, whereas α6ß4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6ß4E presented in "ringlike" patterns measuring ∼1.75 × 0.72 microns and containing actin and CD9 at cell-ECM locations. In contrast, α6ß4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6ß4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions.

Gene Editing of α6 Integrin Inhibits Muscle Invasive Networks and Increases Cell-Cell Biophysical Properties in Prostate Cancer.

Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6ß1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 µmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region.

Cross-validation of murine UV signal transduction pathways in human skin.

Acute UVB irradiation of mouse skin results in activation of phospatidyinositol-3 (PI-3) kinase and mitogen-activated protein kinase (MAPK) pathways leading to altered protein phosphorylation and downstream transcription of genes. We determined whether activation of these pathways also occurs in human skin exposed to 4x minimal erythemic dose of UVB in 23 volunteers. Biopsies were taken prior to, at 30 min, 1 and 24 h post-UVB. In agreement with mouse studies, the earliest UV-induced changes in epidermis were seen in phospho-CREB (two- and five-fold at 30 min and 1 h) and in phospho-MAPKAPK-2 (three-fold at both 30 min and 1 h). At 1 h, phospho-c-JUN and phospho-p38 were increased five- and two-fold, respectively. Moreover, phospho-c-JUN and phospho-p38 were further increased at 24 h (12- and six-fold, respectively). Phospho-GSK-3beta was similarly increased at all time points. Increases in phospho-p53 (12-fold), COX-2 (four-fold), c-FOS (14-fold) and apoptosis were not seen until 24 h. Our data suggest that UVB acts through MAPK p38 and PI-3 kinase with phosphorylation of MAPKAPK-2, CREB, c-JUN, p38, GSK-3beta and p53 leading to marked increases in c-FOS, COX-2 and apoptosis. Validation of murine models in human skin will aid in development of effective skin cancer chemoprevention and prevention strategies.

A new species of Euchaetes Harris from southern Arizona (Erebidae, Arctiinae).

Euchaetesnancyae sp. n. is described from southeastern Arizona. Although superficially similar to species of Pygarctia Grote, structural and molecular variation shows it to be most closely related to Euchaeteshelena (Cassino). Adults, genitalic structure, eggs, and first instar larvae are described and illustrated. The larval host plant remains unknown. Euchaeteshelena is confirmed as occurring in Mexico.