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Supplementary Data from CD151 Regulates Tumorigenesis by Modulating the Communication between Tumor Cells and Endothelium

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., α3β1, α6β1, and α6β4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151(+) and CD151(−) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151(+) tumors was less pronounced or absent in the CD151(−) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both α3β1 and α6β4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer. (Mol Cancer Res 2009;7(6):787–98)

To date, our understanding of how DNA is packaged in the cell nucleus, condensed from chromatin into chromosomes, and organized throughout the cell cycle remains sparse. Three dimensional (3D) ultrastructural imaging is an important tool for unravelling the organizational structure of chromosomes. For large volume 3D imaging of biological samples, serial block-face scanning electron microscopy (SBFSEM) has been applied, whereby ultrastructural information is achieved by analyzing 3D reconstructions acquired from measured data sets. In this review, we summarize the contribution of SBFSEM for obtaining 3D images of chromosomes to investigate their ultrastructure and organization in the cell and its nucleus. Furthermore, this review highlights the potential of SBFSEM for advancing 3D chromosome research.

Embryoid bodies (EBs) are multicellular three-dimensional (3D) aggregates generated from induced pluripotent stem cells (iPSCs) in suspension and serve as useful biological sources for many downstream applications. Imaging of live EBs has been hampered mainly due to the inherent limitations of the imaging techniques applied to date. This study aimed to image human iPSC (hiPSC) derived EBs to obtain their 3D volume, determining size, morphology, and cell viability from day 7 to 14 using Light Sheet Fluorescence Microscopy (LSFM). Furthermore, chromosomal stability was assessed using Multicolor fluorescence in situ hybridization (M-FISH) from day 8 to 14. EB volume increased from day 7 to 13 which, decreased at day 14. From day 7 to 11, the EBs mainly appeared spherical and morphed into an ellipsoidal shape by day 13. All EBs showed varied external morphologies and larger cavities at day 14. The EB karyotype was diploid 46XY at day 8 and exhibited a low level of aneuploidy from day 10 to 14. This study shows that an increase in cell death affects the morphology and chromosomal stability in EBs derived from hiPSC. We demonstrate that the combination of LSFM and M-FISH helps characterize EBs that will assist future stem cell therapies.

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph+) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph+ ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells. Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph+ ALL which will assist in developing novel targeted therapy for Ph+ ALL patients with BCR-ABL1 independent nonmutational resistance.

Background:Multiple myeloma (MM) is a disease of malignant plasma cells (PC) with poor survival. Disease progression and treatment relapse are attributed to MM cancer stem cells (CSCs) and signaling molecules such as MUC1 and XBP1. The prognostic value of the expression of CSC associated biomarkers, MUC1 and XBP1 in MM, has not been explored previouslyMethod:In this study, we determined the immunohistochemical expression of CSC markers (ALDH1, CD117 and CD34), MUC1 and XBP1in 128 MM formalin-fixed paraffin-embedded bone marrow archival blocks. The expression of biomarkers was assessed for association with clinicopathological variables and the overall survival of patients. Descriptive & initial univariate survival analyses were performed using Kaplan Meier curves. Univariate and multivariable analyses were performed using simple and multiple Cox regression models. The results are reported as crude & adjusted hazard ratios with 95% confidence intervals. Results :Expression of ALDH1 and CD117 was found in 51% and 48% of the tumors, respectively. ALDH1 expression was associated with 1.83 years of reduced survival for patients with CD56 negative tumors. MUC1 expression was observed in 62%, whereas XBP1 was expressed in 48% tumors. Combinatorial group analysis of XBP1 and MUC1 stratified patients into two prognostic groups. Cases with tumors negative for expression of MUC1 and XBP1 (XBP1- / MUC1-) were categorized as a good prognostic group with increased survival of 3.42 years compared to cases with tumors expressing both (Worst prognosis, XBP1+/MUC1+). The adjusted hazard ratio showed a four-fold increased risk of mortality associated with the concordant expression of MUC1 and XBP1 in patients who were >65 years of ageConclusion: Concordant expression of MUC1 and XBP1 in MM defines a subset of patients with adverse outcomes. Of these, patients diagnosed at >65 years of age have a significantly higher risk of death.

The high-order structure of mitotic chromosomes remains to be fully elucidated. How nucleosomes compact at various structural levels into a condensed mitotic chromosome is unclear. Cryogenic preservation and imaging have been applied for over three decades, keeping biological structures close to the native in vivo state. Despite being extensively utilized, this field is still wide open for mitotic chromosome research. In this review, we focus specifically on cryogenic efforts for determining the mitotic nanoscale chromatin structures. We describe vitrification methods, current status, and applications of advanced cryo-microscopy including future tools required for resolving the native architecture of these fascinating structures that hold the instructions to life.

In 2017, the University of Hong Kong and the University of California San Diego co-hosted the first Asian meeting of the recently formed Asia Pacific Research Integrity (APRI) network in Hong Kong. Aligned with planning meetings in 2015 and 2016 funded in part by the US Office of Research Integrity (ORI), the Hong Kong meeting was designed by a multi-national planning committee to address pressing challenges in research integrity: improving multi-national communication; exchanging information on managing misconduct investigations; and sharing best practices to promote research integrity. To create a sustainable, robust international partnership to promote research integrity in the region, the purpose of this 2017 meeting was to foster multi-national awareness, understanding, and opportunities for collaboration. The meeting was defined by four objectives that emerged from the previous meetings: (1) Articulate differences as well as areas of common ground; (2) Identify best or recommended practices; (3) Identify opportunities for research or collaboration; and (4) Set an APRI network agenda for coming years. The key anticipated outcome was to advance the conversation surrounding research integrity among academic institutions and regulators in Asian and Pacific Rim nations. This outcome was evidenced by meeting participation, participant satisfaction, and articulation of next steps for the APRI network.

Multiple myeloma (MM) is a heterogeneous disease of the bone marrow (BM). Its association with Epstein-Barr virus (EBV) remains enigmatic. Aim of our study was to determine expression of latent membrane protein 1 (LMP1), aldehyde dehydrogenase 1 (ALDH1), CD117 and their association with 5-year survival in MM patients. Seven percent of cases expressed LMP1 in MM cells with no association with survival. Whereas, LMP1 expression in CD138- non-neoplastic cells was observed in 80% of the cases, conferring a survival advantage of 1.75 years (mean 3.75 ± 0.28, 95% CI 3.19-4.3). LMP1 in CD138- non-neoplastic cells was associated with CD117 expression in MM cells. Combinatorial analysis of LMP1 and CD117 stratified patients into good prognostic group LMP1+/CD117- (mean survival 4.16 ± 0.39 years) and a worst prognostic group; LMP1-/CD117+ (mean survival 1.02 ± 0.29 years). Our study showed that LMP1 expression in CD138- non-neoplastic cells of BM in MM patients confers a survival advantage.

Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.

Background The seroprevalence of human cytomegalovirus (HCMV) infection ranges from 30 to 90 % in developed countries. Reliable estimates of HCMV seroprevalence are not available for Pakistan. This study determined the seroprevalence and sociodemographic factors associated with HCMV infection in adult populations of Karachi, Pakistan. Methods A seroprevalence survey was conducted on 1000 adults, including residents of two semi-urban communities, and visitors to a government and a private hospital. Questionnaire-based interviews were conducted. Sera were analysed for HCMV-specific IgG and IgM. Chi-square or Fisher’s exact test was used for comparing sociodemographic variables against seropositivity of HCMV-IgG or IgM. Multiple logistic regression modeling was performed for IgG seroprevalence and adjusted odds ratios were computed. Results The seroprevalence of HCMV-IgG and IgM was 93.2 and 4.3 % respectively. 95.3 % of individuals who were IgM seropositive were also seropositive for IgG. Around 6 % (15/250) of women of childbearing age remained uninfected and were therefore susceptible to primary infection. HCMV-IgG seroprevalence was associated with being female (p = 0.001), increasing age (p = 0.002) and crowding index (p = 0.003) and also with lower levels of both education (p

A correction to this paper has been published: https://doi.org/10.1007/s12551-020-00767-5 .

Introduction: The t(9;22) (q34;q11) translocation results in the constative active BCR/ABL tyrosine kinase. Der22 involves the Breakpoint Cluster Region (BCR) gene locus with two principal breaks: a. M-bcr, encoding for the p210-BCR/ABL and b. m-bcr, encoding for the p185-BCR/ABL fusion proteins, respectively. BCR/ABL is the oncogenic driver of Chronic Myeloid Leukemia (CML) and 30% of adult Acute Lymphatic Leukemia (ALL). Activated BCR/ABL kinase is responsible for aberrant activation of multiple signaling pathways, such as JAK/STAT, PI3K/AKT and RAS/MAPK which eventually result in leukemic transformation. Successful targeting of BCR/ABL by selective tyrosine kinase inhibitors (TKIs) such as Imatinib, Nilotinib, Dasatinib and Ponatinib are used for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Most patients with CML in the early stage (CML-CP) treated with TKIs have increased overall survival. However, TKIs have not been as effective in patients with CML blast crisis (CML-BC) or Ph+ ALL. Point mutations in the tyrosine kinase domain (TKD) of BCR/ABL have emerged as the predominant cause of acquired resistance. These mutations are observed in up to 80% of patients with CML-BC and Ph+ ALL and in ~ 50% of Imatinib-resistant patients. In the remaining 20-50% of patients the mechanism of resistance to TKIs remains elusive. The aim of this study was to investigate the mechanism of non-mutational resistance in Ph+ ALL. Methods: As models for non-mutational resistance, we used patient derived long term cultures (PDLTCs) from Ph+ ALL patients with different levels of non-mutational drug resistance and the SupB15RT, a Ph+ ALL cell-line rendered resistant by exposure to increasing doses of Imatinib and cross-resistant against all approved ABL Kinase Inhibitors (AKIs). Cell proliferation was assessed by XTT/MTT and trypan blue dye exclusion. Signaling pathway proteins were assessed by Western Blot analysis. Chromosomal karyotyping was undertaken on single cell genomes using multi-color FISH (M-FISH) technology. Mutation analysis on the ABL kinase domain was done by sequencing the heminested PCR products obtained from SupB15-WT and SupB15RT cell-lines. Results: A non-mutational resistance cell line SupB15RT, was developed by exposing SupB15 cells to an increasing concentration of Imatinib over a 3 month period. SupB15RT were able to grow in 10 µM Imatinib. SupB15RT cells were karyotypically and mutationaly identical to SupB15 WT. All approved AKIs and allosteric inhibitors like GNF-2, ABL001 and Crizotinib were unable to inhibit growth of these cells, except for Dasatinib (IC50 40nM), a multi-target kinase inhibitor. Experiments to determine the mode of resistance revealed high level (3 fold) of activation of AKT/mTOR enabling these cells to grow and proliferate. We targeted the AKT/mTOR pathway using BKM-120 (PI3 Kinase inhibitor), BEZ-235 (PI3 Kinase and mTOR pathway) and Trorin1/Torin2 (mTORC1 and mTORC2) and found that Torin-1 and Torin-2 significantly inhibited proliferation of SupB15RT, in a dose dependent manner, with an IC50 of 11-20 nM. As Dasatinib alone inhibited growth of SupB15RT cells at 40-50nm concentrations, we combined Dasatinib with Torin1 and found that the combination of these two compounds had an additive inhibitory effect on cell growth. Following this we examined clinical samples from patients. We used three different Ph+ PDLTCs: a. HP (BCR/ABL negative), b. PH (BCR/ABL positive and responsive to TKIs) and c. BV (BCR/ABL positive and non-mutational resistant to TKIs). Interestingly, we found that AKT/mTOR pathway was activated in BV cells and its proliferation was inhibited by Torin1 with IC-50 of 50nM. Conclusion: Our experiments revealed an additional pathway involved in the evolution of non-mutational resistance in Ph+ ALL which could assist in developing novel targeted therapy for Ph+ ALL patient(s) with non-mutational resistance. Disclosures Ottmann: Celgene: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Fusion Pharma: Honoraria; Pfizer: Honoraria; Roche: Honoraria.

Introduction: Multiple myeloma (MM) is characterized by expansion of neoplastic plasma cells (PCs). Disease presentation is heterogenous with hypercalcemia, renal failure, osteolytic lesions or a combination thereof. The overall survival of patients and their response to treatment has improved considerably however, MM stays incurable. Patients develop treatment resistance and progress to relapse. Disease progression to treatment resistance and relapse are attributed to complex signaling molecules. In vitro experiments on cell lines are pivotal to determine the functional characterization of these molecules leading to identification of potential therapeutic targets. Cell lines have served as instruments for research in varied disciplines including cancer. Currently, majority of the cell lines in cancer research are from European origin and to a lesser extent from Africans, Asians and Hispanic populations. This lacuna of inadequate racial and genetic representation leads to major consequences such as delays in the benefits of precision medicine, drug efficacy and partial understanding of molecular aspects of the disease. The study was undertaken to establish and characterize a cell line from a patient diagnosed with MM. Methods AKU-MY01 was established from a 38-year male patient diagnosed with ISS stage III, kappa light chain myeloma in 2013. Plasma cells were isolated from the BM aspirate using sterile conditions and cultured in RPMI-1640 supplemented with 10% FBS. Conditioned media from urinary bladder carcinoma cell line 5637 was used to provide growth factors. Isolated suspension cells were observed in culture which were subcultured by splitting in a 1:3 ratios. Detailed characterization of AKU-MY01 was undertaken using gene expression analysis, population doubling time (PDT), determination of clonality, fluorescent in situ hybridization, immunocytochemistry (ICC), karyotyping, transwell assay and short tandem repeat (STR) analysis. Results: AKU-MY01 showed expression of ABCG2dim, Oct4, CD38, CD138, CD19, CD20, CD45, CD56 dim, XBP1, MUC1, EBNA1, and monoclonal Lambda light chain. AKU-MY01 exhibited a human diploid karyotype with 46XY and IGH deletions/translocation in 20% of the cells. It has a PDT of 48-53 hrs. and high invasive potential (15.6%) compared to other MM cell lines. Expression of EBNA at mRNA level was further confirmed through fluorescent ICC. The results from Immunofluorescent ICC showed clusters of tumor cells with concordant expression of membranous CD138 and cytosolic and nuclear expression of LMP1. Further, the expression of LMP1 in CD138 positive tumor cells was confirmed in Patient's trephine from whome AKU-MY01 was derived. Conclusion: To the best of our knowledge, AKU-MY01 is the first MM cell line characterized for LMP1 expression in CD138 expressing myeloma cells. Salient features of AKU-MY01 which make it a unique addition to the existing repertoire of MM cell lines are a) Asian origin, b) diploid karyotype and c) expression of EBV protein and mRNA. Further studies using this cell line model may contribute to understand the biology of EBV positive MM cases. Disclosures No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph+), having a (9;22) chromosomal translocation. The BCR/ABL fusion protein is the hallmark of Ph+ leukemia. BCR/ABL is characterized by deregulated and constitutively activated ABL tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases, particularly by altering the natural history of chronic phase (CP) CML and preventing the previously inexorable progression to terminal blast crisis (BC). However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR/ABL inhibitors represent major clinical problems. The most important mechanism of resistance against TKIs is the selection of leukemic clones driven by BCR/ABL harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). The "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of Ponatinib, a multi-target kinase inhibitor. CML and Ph+ ALL, rarely present at diagnosis with a BCR/ABL harboring a resistance mutation to TKI. Resistant clones may be present and only detectable by highly sensitive methods. We have previously shown that the resistance mutations may influence the biology of BCR/ABL and its transformation potential. We therefore hypothesized that the presence of mutations such as the T315I select for a "dormant cell population" which manifests following initial treatments with TKI inhibitors and treatment failure. The aim of this study was to determine whether the ''gatekeeper'' mutation T315I is able to confer biological features to BCR/ABL influencing its leukemogenic potential. We investigated the influence of T315I on the biology of BCR/ABL in CML and Ph+ ALL. We used Ph+ ALL patient derived long term culture (PDLTCs), factor dependent Ba/F3 cells and syngeneic mouse model of BCR/ABL induced CML-like disease. These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. We observed significantly slower proliferation of Ba/F3 cells and PDLTCs expressing BCR/ABL-T315I compared to the native BCR/ABL. This was further confirmed by undertaking mitotic index calculations and colony formation assays on both cell types. Furthermore, the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). We undertook functional studies to determine the putative signaling pathway and found that Ras/Erk1/2 pathway was activated inT315I positive cells. This study may assist towards therapy decisions in patients with CML/Ph+ ALL with a T315I mutation. Disclosures No relevant conflicts of interest to declare.

Introduction: Multiple myeloma (MM) is a plasma cell (PC) disorder of the elderly, affecting predominantly males. Therapeutic advancement during the last decade has improved overall survival. Crosstalk between malignant PCs and the microenvironment is considered to play an important role in the development of resistance to treatment and relapse. Epstein Barr virus (EBV) is the first B-lymphotropic herpes virus identified for its association with human tumors. Reports relating an association of EBV to MM are few and inconsistent. It has been proposed that initiation of viral lytic cycle in EBV infected B cells is preceded by X-Box binding Protein-1 (XBP-1) mediated differentiation, resulting in a subset of EBV expressing infected PCs. Increased incidence of EBV expressing MM cases have been observed in immune-compromised patients. However, the significance of EBV in the bone marrow (BM) of MM patients has not been fully elucidated. The aim of our study was to evaluate prevalence of EBV in association with MUC1, XBP-1, ALDH1, and B-cell lineage markers (CD20, CD45, CD56, and CD138) in bone marrow trephine biopsies of patients with MM and to assess if there was any association with prognosis in a cohort of 151 MM patients at a single tertiary teaching hospital institution in Pakistan. Methodology: Formalin-fixed, paraffin-embedded trephine blocks of 151 MM cases diagnosed from 2007-2015 were evaluated for immunohistochemical (IHC) expression of MUC1, XBP-1, and EBV along with stem cell marker (ALDH1) and B-cell lineage markers (CD20, CD45, CD56, and CD138). Clinicopathologic details and follow-up data were collected from patients' medical records. Data was analyzed on SPSS 19. Five-year survival was estimated by Kaplan Meier curve for patients with follow-up available for 5 years or more (n=75). Results: Mean age at diagnosis was 57 years (range 30-89) with male preponderance (68.2%). Women (60.4%) with MM presented at an earlier age (median age 55 years, range 42-78) and difference in age of presentation was found to be significant (p value 0.04). Bone lesions were identified in 68.9% of patients at presentation. Stage I, II, and III disease was identified in 32.4%, 23.4%, and 44.1 % of patients, respectively (ISS staging system). Relapse was observed in 79.6% of patients and was significantly correlated with the expression of XBP-1 (p value 0.035). CD56 was expressed in 60.3% of cases and was significantly associated with cases diagnosed after the age of 57 years (p value 0.003). Expression of MUC1 was observed in 62.3% of tumors and 70% of MUC1-positive tumors also expressed XBP-1 (p value 0.06). Although the number of cases expressing EBV in the MM tumor cell population was only 7.2%, we detected EBV in 80.2% of the cases in the adjacent non-neoplastic cells with conspicuous expression in megakaryocytes. Five-year survival analysis (n=75) showed that expression of EBV+ in non-neoplastic cells was associated with significantly improved outcome with an advantage of 1.8 years (95% CI 3.32-4.4) as compared to EBV- cases (p value < 0.005). Among the XBP-1+ tumors, lack of EBV expression in non-neoplastic cells significantly correlated with adverse outcome (95%CI, 0.70-2.6) (p value < 0.005). Conclusions: To the best of our knowledge, this is the first study reporting expression of EBV in non-neoplastic cell population in BM trephines of MM patients. Salient conclusions of our study are as follows: 1. Expression of EBV in the surrounding tumor microenvironment of trephine biopsies of patients with MM is associated with a significant survival advantage; 2. Patients with MM in Pakistan present at a comparatively earlier age; 3. XBP1 expression was associated with relapse. Citation Format: Sheerien Rajput, Khurram Minhas, Iqbal Azam, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Expression of EBV in the non-neoplastic cell population in trephine biopsies of patients with multiple myeloma is associated with a significant survival advantage [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 53.

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., α3β1, α6β1, and α6β4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151(+) and CD151(−) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151(+) tumors was less pronounced or absent in the CD151(−) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both α3β1 and α6β4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer. (Mol Cancer Res 2009;7(6):787–98)

OBJECTIVE: To assess the hormonal effects of Fem7® (Merck, KGaA, Darmstadt, Germany) 100 µg transdermal oestrogen patches on men undergoing first-line androgen-deprivation therapy for prostate cancer. PATIENTS AND METHODS: PATCH is a multicentre, randomized, phase II trial for men with locally advanced or metastatic prostate cancer, comparing luteinizing hormone-releasing hormone agonist therapy with oestrogen patches. To assess the dosing schedule for the patches, as this was the first time that this brand of patch had been used in men, and to reassure patients and participating clinicians, the Independent Data Monitoring Committee agreed to early release of hormonal data from this study. RESULTS: Oestradiol, testosterone and prostate-specific antigen (PSA) levels are presented for the first group of 14 patients who received the patches (with 1 withdrawal) and for whom there were ≥12 weeks of follow-up by March 2007. After 12 weeks, testosterone levels (nmol/L) in eight of the 13 patients were 2. The median (range) serum oestradiol levels was 442 (52.1–1542) pmol/L and all patients had a PSA response, with eight having a PSA level of

To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.

OBJECTIVE To report the influence of transdermal oestradiol therapy on the vascular dynamics of men with advanced prostate cancer. PATIENTS AND METHODS Twenty patients with newly diagnosed locally advanced or metastatic prostate cancer (10 each) were treated using transdermal oestradiol patches. The vascular flow was assessed 6-monthly before and during a year of therapy using arterial and venous Doppler and duplex ultrasonography, arterial and venous photoplethysmography and opto-electronic plethysmography. RESULTS Arterial flow, as measured by the mean and peak systolic velocities and photoplethysmography, significantly increased over time. Arterial compliance initially decreased but had normalized after 12 months. The venous variables were unaffected. As a result, the total limb blood flow and the capillary filtration rate were significantly increased. CONCLUSION Transdermal oestradiol therapy causes an increase in arterial but not venous flow, and an initial decrease in arterial compliance, which adapts to the physiological range with time. It is possible that these changes may account for the increase in cardiovascular toxicity seen in the early phase of oestrogen therapy, and the cardioprotective effect that accrues thereafter.

Background & Aims: The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency. Methods: TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry. Results: On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach.

The effect of toremifene on P-glycoprotein-mediated multidrug resistance (MDR) in breast and head and neck cancer cell lines was measured in vitro and in vivo. Pgp expression was low and high, respectively, in drug-sensitive (MCF7-S, KB) and drug-resistant (MCF7-R, MCF7-R1, KBV1) cell lines. Toremifene (7.5 microM) significantly enhanced cytoplasmic and nuclear accumulation of doxorubicin in drug-resistant cells. Toremifene (10 microM) increased the in vitro cytotoxicity of doxorubicin in drug-resistant breast cancer cells (13-fold and 21-fold for MCF7-R and MCF7-R1, respectively) without affecting the sensitivity of MCF7-S cells. Similarly, toremifene (10 microM) caused a 12-fold increase in the sensitivity of KBV1 cells to vinblastine. In contrast, toremifene (5 microM) reduced the net uptake of the radiolabelled Pgp substrate, Tc-99m-sestamibi, in the Pgp-overexpressing cell lines by factors of 0.32 and 0.42 for MCF7-R1 and KBV1 cells, respectively (p0.01), and, to a lesser extent, by corresponding factors of 0.89 and 0.86 in the drug-sensitive cell lines (p0.05 and p0.05, respectively). In nude mice bearing both KB and KBV1 xenograft tumours, significantly higher tumour levels of Tc-99m-sestamibi were recorded in KB tumours compared with KBV1 tumours. After 3 days of treatment with intraperitoneal toremifene (25 mg/kg), tumour levels of Tc-99m-sestamibi were reduced in KB and KBV1 tumours but only statistically significantly for KB tumours. Toremifene is a potent MDR modulating agent with respect to chemotherapeutic agents but has the opposite effect with respect to Tc-99m-sestamibi. This finding is of importance in view of the widespread use of Tc-99m-sestamibi as an imaging surrogate for a chemotherapeutic agent.

Trefoil factor family (TFF) domain peptides, products of mucin-secreting epithelial cells, are thought to influence mucosal integrity. Molecular studies revealed that mammalian TFFs lack transmembrane domains. Using immunocytochemistry and FACS analysis we demonstrated the association of TFF1 with the cell membrane in MCF-7 (a breast adenocarcinoma cell line), and tested the hypothesis that glycosylphosphatidylinositol (GPI) linkage is the mechanism for this association. Cleavage of GPI anchorage using phospholipase C did not affect TFF1 binding to the cell membrane. Our results demonstrate for the first time that TFF1 is associated with the cell membrane of MCF-7 cells and is not linked via a GPI anchor.

We have recently demonstrated that human TFF2 inhibits apoptosis in the non-TFF2 expressing breast adenocarcinoma cell line MCF-7. In this study we examined the impact of TFF2 and an anti-TFF2 antibody (hSP3) on the survival of other human adenocarcinoma cell lines ; TFF2-positive (LS174T and SW480) and TFF2-negative (MCF-7 and T47D). Addition of TFF2 protected the (TFF2– ) lines but had no effect on those constitutively expressing TFF2. Blocking with hSP3 significantly increased apoptosis in the (TFF2+) cell lines with minimal effect on the (TFF2– ) cells. Our results show that the cytoprotective effect of TFF2 seen in MCF-7 cells is not cell line-specific and can be abrogated by inhibition of its expression.

Androgen independence is the major cause of endocrine therapy failure in advanced prostate cancer (PC). To examine the effects of human androgen receptor (AR) expression on growth of human PC cells, transfection of full-length AR cDNA in an androgen-insensitive human prostatic adenocarcinoma cell line (DU145) was performed. Transcriptional activity of AR was confirmed by the MMTV luciferase assay and AR expression was assessed by reverse transcriptase polymerase chain reaction, Western blotting, and immunocytochemistry. Two stable transfectant cell lines expressing functional AR were established and passaged over 60 times. Under standard culture conditions, AR expression in transfected cells was predominantly cytoplasmic. Exposure to dihydrotestosterone (DHT; 60 pM-10 nM) resulted in a rapid (maximal at 30 minutes) translocation of AR to the nucleus. Treatment with DHT (5 nM) caused a significant reduction in cell-cell adhesion and aggregation accompanied by a decrease in E-cadherin expression. This was associated with up to 40% inhibition of proliferation and approximately two-fold increase in apoptosis. These results suggest that gene transfer-mediated AR expression in DU145 cells confers sensitivity to DHT, modulates cell-cell adhesion through E-cadherin, and suppresses cell growth by inhibiting proliferation and promoting apoptosis. This provides a model for studies of AR-regulated cell signalling and identification of novel androgenregulated genes in PC.

Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per-operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol-fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the ‘lifetime’ factor τ. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger τ (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean τ of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy-associated stroma (p < 0.05). Also, benign collagen had a longer τ than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy-associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy-associated stroma were obtained even with small patient numbers, indicating that lifetime-based instruments can be developed for real-time diagnostic imaging with microscopic resolution. Copyright  2003 John Wiley & Sons, Ltd.

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.

This study examines the coexpression of MUC1 mucin and trefoil factor 1 (TFF1) and their relationship to progression of renal cell carcinoma (RCC). Immunohistochemistry was performed on tumor and adjacent normal tissue from clear-cell RCC (n = 60) and tissues from normal controls (n = 5) using a set of well-characterized monoclonal antibodies recognizing different epitopes of MUC1 and TFF1. Results of immunohistochemistry were compared with clinical parameters, including tumor grade, tumor size, presence of metastasis, and progression-free survival of patients after surgery. In normal tissue, MUC1 and TFF1 were absent from the normal proximal tubular epithelium but were identified in distal and collecting tubular epithelium. In RCC, increased MUC1 expression positively correlated to tumor progression. MUC1 recognized by HMFG1 was associated with large tumor size (P < .05), distant metastasis (P < .05), and invasion of large veins (P < .05). Expression of the under-glycosylated form of MUC1 recognized by SM3 was found to correlate to time to progression (recurrence, metastasis, or death of patient; P < .001). Expression of TFF1 did not significantly correlate with any prognostic parameters. However, there was a significant correlation (P < .01) between TFF1 and MUC1 expression (HMFG2 epitope) in RCCs. These results are consistent with the following conclusions: (1) MUC1 may be an independent prognostic marker in RCC; (2) TFF1 is frequently coexpressed with MUC1 and may act synergistically; and (3) RCC may originate from distal tubular epithelium.

Introduction: Androgen receptor (AR) expression has been shown to correlate with improved survival in luminal A, B and triple negative (TN) breast cancer (BCa). Cancer stem cells (CSC) with CD44+/CD24- and ALDH1+ phenotype is associated with pro-invasive gene signature in BCa. Aim of our study was to evaluate prognostic significance of AR expression in relation to CD44, CD24 and ALDH1 in invasive BCa. Methodology: Immunohistochemical expression of AR, CD44, CD24 and ALDH1 was evaluated in 180 FFPE blocks of BCa cases from one institution. Clinico-pathological details and follow-up data were collected from medical charts. Data was analyzed on SPSS 19. Five-year survival was estimated by Kaplan Meier. Results: Median age of patients was 55 years (range: 28-87). Fifteen percent of patients were diagnosed with stage I, 52.2% with stage II, 25% with stage III and 7.8% with stage IV disease. Mean duration of follow-up was 4.5 years (SD+ 2.7) and 29% mortalities were attributed to BCa. AR was expressed in 63.9%, ER in 61%, PR in 51% & HER-2/neu in 30% of tumors. CD44, CD24 and ALDH1 were expressed in 60%, 43.3% & 28.3% of tumors respectively. AR expression was associated (p-value Five year survival of patients with AR+ versus AR- expressing tumors, showed that patients with AR+ tumors had significantly better outcome (p-value=0.03) with survival advantage of 39.6 months. Survival advantage decreased by 9.6 months in tumors expressing CD44+/CD24-/AR- (p-value=0.01) phenotype and by 12 months in patients whose tumors were phenotypically ALDHI+ /AR- (p-value=0.12). We stratified TN tumors into 2 groups: A. TN/CD24+/AR+; B. TN/CD24-/AR- and found that group A tumors had a trend towards better 5 survival (p-value=0.09). Conclusions: Three individual markers (CD44, CD24, AR) may afford prognostic information. Combinations of CD44, CD24 and AR permitted patient stratification into 3 risk categories: poor risk CD24-/AR-; intermediate risk CD44+/CD24-/AR- and good risk CD24+/AR+. Stratification of TN group showed that loss of AR and CD24 was associated with poorest outcome. These results underscore the relevance and importance of AR and CD24 as prognostic markers in invasive BCa. Citation Format: Nazia Riaz, Romana Idress, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Expression of androgen receptor and CD24 correlates with improved 5-year survival in Pakistani patients with invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2017-3822

Introduction: Metaplastic carcinoma (MCa) is a rare and aggressive subtype of invasive breast carcinoma exhibiting epithelial to mesenchymal transition (EMT) where tumor of epithelial origin manifests differentiation into non-glandular, mesenchymal phenotypes such as spindloid, chondroid or osseous components. Acquisition of EMT is associated with low expression of e-cadherin, claudins and high expression of vimentin. Biological mechanisms for acquisition of metaplastic components within MCa are not well understood. The present study was undertaken to establish and characterize a cell line from a patient diagnosed with MCa and to evaluate the role of mesenchymal stem cells (MSC) in attainment of metaplastic phenotype. Methodology: AKU-BC-42 was established from a 65 years old Pakistani female patient diagnosed with T4N1M0 MCa. Specimen was procured and processed under sterile conditions and cultured in DMEM supplemented with 10% FBS. Epitheloid colonies were visualized after 3 weeks of culture, which were passaged and propagated. AKU-BC-42 was phenotypically and genotypically characterized using karyotyping, gene expression analysis, immunocytochemistry and florescent insitu hybridization. MSC marker expression was assessed by flow-cytometry. AKU-BC-42 was cultured under appropriate conditions for assessment of differentiation along osteogenic, adipogenic and chondrogenic pathways. Lineage differentiation was evaluated by special immunohistochemical stains and induction of lineage differentiation genes was assessed by Q-PCR. Results: AKU-BC-42 was found to have a human karyotype with multiploidy and population doubling time of 60 hours. Gene expression profiling revealed negative expression for estrogen and progesterone receptors and positive expression of androgen receptor (AR) and HER-2/neu. FISH analysis was negative for HER-2/neu amplification. Basal (5, 14 & 19) and luminal cytokeratins (8 & 18) were expressed at mRNA level along with myoepithelial markers (CD10, S100A7, p-cadherin, desmin, S100A4, S100A2 & á-SMA). AKU-BC-42 demonstrated MSC pool with expression of CD73 (79.2%), CD90 (10.3%) and CD105 (30.2%) as revealed by flow-cytometry. Osteogenic differentiation was assessed by von kossa stain for mineralization with up-regulation of ALPL and OPN genes. Similarly, differentiation of AKU-BC-42 into mature adipocytes was evaluated with Oil red O staining of lipid droplets and expression of FABP4 gene. Chondrogenic induction was achieved by performing pellet cultures. Collagen synthesis in extracellular matrix of chondrogenic beads was assessed by mason trichrome staining with up-regulation of ACAN, COL10A1 and COMP. Conclusions: We report a novel MCa cell line containing a MSC pool. MSC pool within MCa may be responsible for acquisition of metaplasia in this rare subtype of breast cancer. Further studies on this cell line may reveal the biological mechanisms of MCa of the breast. Citation Format: Nazia Riaz, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Role of mesenchymal stem cells in acquisition of metaplasia in AKU-BC42 breast metaplastic carcinoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5802. doi:10.1158/1538-7445.AM2017-5802

Objective To assess the level and morphological distribution of cyclooxygenase (COX)-1 and -2 in human prostates and to determine any association with the Gleason grade of prostate cancer. Materials and methods The study comprised 30 samples from patients with benign prostatic hyperplasia (BPH) and 82 with prostate cancer. Immunohistochemistry was used to assess the expression of COX-1 and -2, and 13 samples were also assessed using immunoblotting (six BPH and seven cancers). Results For both BPH and prostate cancer, COX-1 expression was primarily in the fibromuscular stroma, with variable weak cytoplasmic expression in glandular/neoplastic epithelial cells. In contrast, COX-2 expression differed markedly between BPH and cancer. In BPH there was membranous expression of COX-2 in luminal glandular cells and no stromal expression. In cancer the stromal expression of COX-2 was unaltered, but expression by tumour cells was significantly greater (P = 0.008), with a change in the staining pattern from membranous to cytoplasmic (P

Objective To analyse and compare the expression of cyclooxygenase (COX) enzymes in schistosoma-associated bladder cancer, and to determine any association with tumour grade or stage. Materials and methods Sixty paired samples of tumour and adjacent nonmalignant urothelium were identified. There were 25 squamous and 28 transitional cell carcinomas, and seven adenocarcinomas. Serial sections were obtained and a standard three-layer immunohistochemistry protocol, using COX-1- and COX-2-specific mouse monoclonal antibodies, applied. Results COX-1 was expressed mostly in nonvascular smooth muscle with weak reactivity in malignant and nonmalignant urothelium. Nonmalignant urothelium expressed COX-2 weakly, notably in areas of dysplasia and squamous metaplasia whereas there was a significant increase in COX-2 (P

Human MUC1 mucin is a high-molecular-weight transmembrane glycoprotein, which is apically expressed in the majority of glandular epithelia. During embryonic development, changes in the pattern of MUC1 mucin expression coincide with the onset of glandular differentiation. This mucin is also frequently overexpressed and aberrantly glycosylated in carcinomas. To investigate the potential role of MUC1 mucin in morphogenesis, a full length MUC1 cDNA was transfected into murine mammary adenocarcinoma (410.4) and Madin-Darby canine kidney (MDCK) cells. This generated four clonal cell lines. Western blotting, FACS analysis, and immunohistochemistry confirmed expression of MUC1. All four MUC1-expressing clones demonstrated altered morphogenesis when cultured in three-dimensional type I collagen gels. While parental and vector control 410.4 cells formed compact spherical structures, the MUC1-expressing clones formed complex branching structures. Similarly, while parental and vector control MDCK cells formed small circumscribed colonies with a central lumen, the MUC1-expressing clones formed elongated tubules. MUC1 expression was also associated with reduced cellular cohesion and enhanced migration on type I collagen-coated surfaces for all except one of the clones, which expressed only low levels of MUC1 on the cell surface. These results show that MUC1 expression stimulates morphogenetic changes in two distinct epithelial cell lines. Taken together with previous observations on MUC1 expression in embryonic development and carcinomas, this finding suggests that MUC1 may induce changes in tissue architecture in both normal development and cancer.

Functional overexpression of Bcl-2 has been reported to confer an anti-apoptotic potential in a variety of cell types. The role of Bcl-2 in epithelial cell-cycle control and in interactions with other cell-cycle regulators is not clearly understood. Its expression has been correlated with the hormono- and chemo-resistant phenotype in advanced prostate cancer. The aim of this study was to investigate the mechanisms through which Bcl-2 mediates increased cytotoxic chemoresistance by assessing alterations in the expression of cell death regulatory molecules. The DU145 human prostatic adenocarcinoma cell line was stably transfected with a Bcl-2 encoding expression plasmid. Two Bcl-2 transfectants, DKC9 and DKC11, were expanded for further study. The effects of Bcl-2 expression on cellular proliferation, cell death (+/- adriamycin or thapsigargin), and expression of cell-cycle/death regulators (p53, PCNA, Bax, Bak, Bcl-X(L)) were evaluated. Compared with controls, Bcl-2 transfectants showed no difference in the rate of proliferation, a decrease in p53 (approximately two-fold), an increase in Bax (approximately two-fold) and PCNA (approximately three-fold), and no change in the levels of Bcl-X(L) and Bak proteins. DKC9 and DKC11 also exhibited a significantly increased chemoresistance to adriamycin (0.0025-5 microM) and thapsigargin (0.0025-5 microM) compared with controls. In the presence of thapsigargin or adriamycin, levels of Bcl-2 and its heterodimeric partner Bax were elevated approximately two-fold with no change in Bak in Bcl-2 transfectants in contrast to controls, where Bak was increased (two-fold). This is the first study to demonstrate that Bcl-2 transfection modulates the expression of mutant p53, Bax, and PCNA in prostate cancer cells. Moreover, Bcl-2 overexpression conferred a significant cytotoxic chemoresistance and altered the balance of expression of death promoters (from Bak, a dominant death promoter in controls, to Bax) in response to thapsigargin and adriamycin.

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.

A comparison has been made of the phenotypic expression of MHC class I antigens with the corresponding HLA-A genotype in 15 cases of benign prostatic hyperplasia (BPH) and 34 cases of primary locally invasive prostatic carcinoma. Expression of class 1 protein, detected by immunocytochemistry, was partially or completely lost in approximately 90% of the tumours examined. Comparative genomic analysis of the beta2 microglobulin (beta2m) gene and 15 individual HLA-A haplotypes using a polymerase chain reaction (PCR)-based method demonstrated abnormal gene dosage in the minority of cases: homozygous deletion of the beta2m locus was detected in one case and HLA-A allele in two cases (HLA-A1 and HLA-A2, respectively), representing approximately 8% of the population studied. This first comparative study of gene dosage and expression of class 1 protein reported for prostate cancer reveals that deletion is not the cause of the partial or complete loss seen in the majority of cases. This raises the possibility, in the future, for novel selective immunomodulatory therapeutic strategies which stimulate a clinically significant re-expression of class 1 protein and associated cytotoxic T-cell response.

AIM: To investigate the correlation between androgen receptor expression and fibroblast growth factor 8 (FGF8) mRNA levels. METHODS: 39 human prostate cancers and 14 benign prostatic hypertrophy specimens were examined immunohistochemically for androgen receptor expression and by in situ hybridisation and reverse transcription polymerase chain reaction for FGF8 expression. RESULTS: In 39 tumours there was a statistically significant negative correlation between tumour grade and FGF8 expression and a positive correlation between FGF8 and androgen receptor expression. All 14 benign hypertrophy specimens expressed moderate to high levels of FGF8 and androgen receptor. CONCLUSIONS: Loss of FGF8 may be a factor involved in the development of prostatic cancer.