High Proliferative State Research Articles

TP53 Aberrant B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Are Uniquely Sensitive to a Combination of Pyrimidine De Novo Synthesis Blockade and Replication Checkpoint Inhibition

Background Unlike most solid tumors, the incidence of TP53 aberrations in ALL is only 3% at diagnosis. However, in relapsed ALL this number increases to 12%. More importantly, at relapse, loss of p53 function is associated with therapy failure, with an overall survival of only 20%. Hence, there is a clear clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Owing to their high proliferative state, leukemic blasts show a high dependency on pyrimidines for DNA synthesis. A lack of pyrimidines leads to replication stress, causing activation of p53 and the DNA damage response pathway. Results We used CRISPR/Cas9 to generate TP53 deficient variants of the TP53 proficient BCP-ALL cell lines Nalm6 and RCH-ACV. We used these cell lines in a CRISPR screens and high throughput drug screen, and identified a strong synergy between the targeting of pyrimidine synthesis and DNA checkpoint kinase ATR and its downstream effectors Chk1 and Wee1 in TP53 deficient ALL. To validate these results, we tested the efficacy of a checkpoint inhibition together with Orludodstat (BAY-2402234), an inhibitor of Dihydroorotate Dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis. This clinical grade inhibitor was evaluated in either wild type or TP53 deficient BCP-ALL and T-ALL cell line models and patient derived xenografts (PDX). A strong apoptotic response was observed when Orludodstat was combined with the ATR inhibitor Berzosertib, most notably in TP53 deficient ALL. This p53 dependency was confirmed using ex vivo cultures of patient derived xenografts (PDX) of diagnosis and relapse pairs, where each diagnostic sample was wild type for TP53, and the relapse carried a TP53 aberration. While little or no synergy was observed in the diagnostic samples, a strong efficacy of the combination was observed in the matched TP53 deficient relapsed samples (Figure 1 A). Preliminary results from i n vivo experiments using p53 deficient patient derived xenografts indicate that the combination of Orludodstat and Berzosertib is well tolerated, while significantly delaying leukemic growth (Figure 1 B). Conclusion Together, our findings suggest that the combined targeting of de novo pyrimidine synthesis and ATR signaling may present a specific and non-genotoxic vulnerability for TP53 aberrant ALL.

Abstract 2978: E2F1 regulates cell competition, increase proliferation and inhibits chemotherapy resistance in cancer cells

Abstract Introduction: Tumor heterogeneity is a key element during the development of chemo-resistance and tumor relapse. It remains unknown how resistant tumor cells influence sensitive cells during conditions of cohabitation. The goal of our study was to determine driving factors mediating interactions between resistant and sensitive cancer cells and the effects of cohabitation on both phenotypes. Materials and methods: We used two isogenic ovarian cancer cell lines pairs, sensitive and resistant to platinum: OVCAR5 (IC50 = 2.05 uM) vs. OVCAR5-CisR (IC50 = 7.26 uM) and PEO1 (IC50 = 5.92 uM) vs. PE04 (IC50 = 14.45 uM) labeled with either GFP or RFP. The cell pairs were seeded together in long direct term culture, separated by fluorescence activated cell sorting (FACS), and used for RNAseq analysis, proliferation assays, Cisplatin IC50 determinations, and cell cycle analysis. Results: Long Term direct co-culture (14 Days) of platinum-sensitive and -resistant cells induced increased proliferation (p < 0.001) and a greater proportions of cells in the S and G2 phases of the cell cycle in sensitive PEO1 and OVCAR5 cells compared with cells cultured by themselves. Resistance to platinum of the sensitive cell lines decreased further by co-culture (IC50 change from 5.92 uM to 2.79 uM in PE01, and from 2.05 uM to 1.51 uM in OVCAR5). No changes in cell proliferation, or resistance to platinum occurred in the platinum resistant cells. Use of conditioned media of resistance cells did not change proliferation, or response to platinum of sensitive cells. RNAseq detected 3840 differentially expressed genes; 1774 upregulated and 2066 downregulated. IPA analysis showed enrichment of the cell cycle pathways (Cell Cycle Control, Cyclins and Cell Cycle Regulation, among others) and upregulation of cell proliferation genes including the E2F1 transcription factor. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured platinum sensitive cells vs cells in monoculture. Furthermore, western blot measurements showed increased levels of other proteins involved in cell cycle regulation such as CDK4 and CDK6. Cell cycle analysis indicated an increase of cells in S and G2 phase in the co-cultured platinum sensitive cells (18.8% up G2 phase; p = 0.031). Treatment with the CDK4/6 inhibitor Palbociclib or with the E2F1 inhibitor HLM006474 reverted the increase in proliferation rate. Conclusion: Long term cohabitation of platinum-sensitive and -resistant cancer cells drives sensitive cells to a higher proliferative state, which is more responsive to platinum. Direct cell-cell contact modulates cell cycle progression in sensitive cells through a mechanism regulated by E2F1. Our data reveal unexpected effects caused by interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors. Citation Format: Andres Felipe Valdivia, Matthew Cowan, Guangyuan Zhao, Horacio Cardenas, Daniela Matei. E2F1 regulates cell competition, increase proliferation and inhibits chemotherapy resistance in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2978.

Energetic metabolic reprogramming in Jurkat DFF40-deficient cancer cells.

DNA fragmentation factor 40 (DFF40), or the caspase-activated DNase (CAD), is an endonuclease specific for double-stranded DNA. Alterations in its function and expression have been linked to apoptosis resistance, a mechanism likely used by cancer cells. However, how the DFF40-related apoptosis resistance pathway occurs remains unclear. Here, we sought to determine if DFF40 expression could be linked to cell metabolism through the regulation of mitochondrial integrity and function. We demonstrated that DFF40-deficient cells are more resistant to staurosporine and tributyltin (TBT)-induced apoptosis, and express higher levels of Mcl-1 at basal state. Treatment with TBT induces higher Bcl-2 and caspase-9 mRNA transcripts in DFF40 KO Jurkat cells, as well as enhanced Bcl-2 phosphorylation. A loss of DFF40 expression induces a higher mitochondrial mass, mtDNA copy number, mitochondrial membrane potential, and glycolysis rates in resting T cells. DFF40-deficient cells exhibit the Warburg effect phenotype, where they rely significantly more on glycolysis than oxidative phosphorylation and have a higher proliferative state, demonstrated by a higher Ki-67 transcription factor expression and AKT phosphorylation. Finally, we demonstrated with cell fractioning that DFF40 can translocate to the mitochondria following apoptosis induction. Our study reveals that DFF40 may act as a regulator of mitochondria during cell death and its loss could compromise mitochondrial integrity and cause an energetic reprogramming in pathologies such as cancer.

Abstract A58: Transcription factor SREBP2 mediates ovarian cancer drug resistance and recurrence

Abstract Ovarian cancer, the most lethal gynecologic malignancy, has a poor prognosis. Existing chemotherapies are challenged by the emergence of drug resistance and metastasis of tumor cells, which obstruct sustained, relapse-free patient cures. Resistance can emerge from pre-existing mutations, secondary mutations, or other nongenetic mechanisms or metabolic adaptations. SREBPs are necessary for cell survival in low-nutrient conditions. SREBP1 predominantly regulates fatty acid and glycolysis while SREBP2 regulates cholesterol metabolism. SREBP2 is a sterol sensitive transcription factor regulating cholesterol homeostasis including the expression of LDLR and HMGCR. Recent reports suggest that SREBP2 may promote mesenchymal and stem-cell states. These observations support our working hypothesis that the regulation of cholesterol and fatty acids through SREBP2 is critical for drug tolerance, resistance, and viability of a cancer stem cell population. We further hypothesize that drug-tolerant ovarian cancer cells depend on SREBP2 to re-enter the cell cycle and proliferate. To test this hypothesis, we assessed the importance of SREBP2 in an in vitro drug tolerant model. Ovarian cancer cells were treated with high concentrations of paclitaxel for 1 week and the surviving cells were examined. SREBP2, HMGCR, and related factors were significantly upregulated in these surviving drug-tolerant cells. To evaluate the function of SREBP2 in drug-tolerant cells, stable cell lines targeting the SREBF2 gene using CRISPR technology were created in OVCAR8. Using this SREBF2-KD line, we assessed the response of ovarian cancer cells to drug treatment under different environments. SREBF2-KD cells were more sensitive to statin and paclitaxel in low-sterol environments compared to control cells. These observations highlight the need for SREBP2 in stressed conditions for cancer cells to survive drug treatment. Moreover, after treatment with high concentrations of paclitaxel, SREBF2-KD cells grew back significantly slower compared to control cells. To characterize the mechanisms controlled by SREBP2, we examined the effects on the cell cycle. We have observed a significant change in the distribution of the cell cycle phases between SREBF2-KD and control cells treated with paclitaxel, which suggests an important role for SREBP2 in ovarian cancer cells’ exit from cell cycle arrest after chemotherapy. To further examine the role of SREBP2 in drug resistance and tumor relapse, we are currently extending these in vitro observations to in vivo models. In summary, our study illustrates the crucial role of cholesterol pathway for ovarian cancer not only under stress conditions such as chemotherapy, but also in return to a high proliferative state upon recurrence. These data suggest that targeting SREBP2 may represent a new approach for sequential therapy to eliminate cancer cells that survive initial treatment. Citation Format: Galina Karashchuk, Alexander S. Brodsky. Transcription factor SREBP2 mediates ovarian cancer drug resistance and recurrence. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A58.

Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development

Leiomyosarcoma is a rare malignant smooth muscle tumor which can be very unpredictable. Myosin II is involved in many functions, including cell contraction, migration, and adhesion. The phosphorylation of myosin regulatory light chain (MLC) by myosin light chain kinase (MLCK) determines the activity of Myosin II. However, it is still unclear whether MLC phosphorylation is involved in cell proliferation in leiomyosarcoma. In this study, we aimed to explore the role of MLCK-dependent MLC phosphorylation in leiomyosarcoma development. We found that the expression of MLCK, phosphorylated MLC, and Ki67 in leiomyosarcoma was significantly higher than in leiomyoma and adjacent normal smooth muscle cells. MLCK expression was significantly correlated with phosphorylated MLC level. Kaplan-Meier survival analysis revealed that patients with high expression of MLCK or phosphorylated MLC had shorter overall survival times compared with the patients with low expression of MLCK or phosphorylated MLC. In vitro studies revealed a causative link between MLC phosphorylation and cellular proliferation as expression of phosphomimetic MLC (T19D, S20D) increased cellular proliferation as assessed by Ki67 staining. In contrast, MLCK specific inhibitor reduced cellular proliferation. We concluded that MLCK, phosphorylated MLC and Ki67 were overexpressed in leiomyosarcoma. MLCK dependent MLC phosphorylation might be responsible for the high proliferative state in leiomyosarcoma. MLCK and phosphorylated MLC are potential prognostic indicators of leiomyosarcoma.

The In Vitro Influence of a Genetic Superoxide-Hydrogen Peroxide Imbalance on Immunosenescence.

As superoxide is a key molecule of inflammatory activation, superoxide-hydrogen peroxide (S-HP) imbalance genetically caused could alter immunosenescence patterns. To test this hypothesis, we collected and cultured peripheral blood mononuclear cells (PBMCs) carrier's different genotypes of a genetic polymorphism located in the superoxide dismutase manganese-dependent gene (Val16Ala-SOD2). We used an in vitro genetic model based on previous studies, which suggested an association between homozygous genotypes (AA and VV) and alterations in oxidative-inflammatory mediators. PBMCs collected from young healthy volunteers were cultured in the presence of phytohemagglutinin, as well as the following cell culture passages obtained from the 72-hour initial culture. Each follow passage started with the same cell concentration (1 × 105 cells). The general immunosenescence pattern was observed independent of SOD2 genotypes: cellular proliferation until the 15th passage, when cellular arrestment occurred in the G0/G1 phase. From the 10th passage, a higher proliferative state was observed, indicating inflammatory hyperactivation, with an increase in the levels of inflammatory cytokines (IL-1, IL-6, and TNFα), nitric oxide, superoxide, lipoperoxidation, protein carbonylation, reactive oxygen species, and DNA damage. The S-HP imbalance affected the intensity of some immunosenescence parameters. AA cells, which present basal high HP levels, were associated with higher DNA damage and lipoperoxidation levels, whereas VV, which present basal high S levels, was associated with higher proinflammatory cytokine levels. In summary, the results suggested that a basal S-HP imbalance could affect the intensity of some immunosenescence markers, and this influence could explain the potential association between an imbalance of genotypes (AA and VV) and the risk of developing some chronic diseases.

Constitutive β-catenin activation in osteoblasts impairs terminal osteoblast differentiation and bone quality

Accumulating evidence suggests that Wnt/β-catenin signaling plays a central role in controlling bone mass. We previously reported that constitutive activation of β-catenin (CA-β-catenin) in osteoblasts potentially has side effects on the bone growth and bone remodeling process, although it could increase bone mass. The present study aimed to observe the effects of osteoblastic CA-β-catenin on bone quality and to investigate possible mechanisms of these effects. It was found that CA-β-catenin mice exhibited lower mineralization levels and disorganized collagen in long bones as confirmed by von Kossa staining and sirius red staining, respectively. Also, bone strength decreased significantly in CA-β-catenin mice. Then the effect of CA-β-catenin on biological functions of osteoblasts were investigated and it was found that the expression levels of osteocalcin, a marker for the late differentiation of osteoblasts, decreased in CA-β-catenin mice, while the expression levels of osterix and alkaline phosphatase, two markers for the early differentiation of osteoblasts, increased in CA-β-catenin mice. Furthermore, higher proliferation rate were revealed in osteoblasts that were isolated from CA-β-catenin mice. The Real-time PCR and western blot examination found that the expression level of c-myc and cyclin D1, two G1 progression-related molecules, increased in osteoblasts that were isolated from the CA-β-catenin mice, and the expression levels of CDK14 and cyclin Y, two mitotic-related molecules that can accelerate cells entering into S and G2/M phases, increased in osteoblasts that were isolated from the CA-β-catenin mice. In summary, osteoblastic CA-β-catenin kept osteoblasts in high proliferative state and impaired the terminal osteoblast differentiation, and this led to changed bone structure and decreased bone strength.

SNS01-T Modulation of eIF5A Inhibits B-cell Cancer Progression and Synergizes With Bortezomib and Lenalidomide

The high rates of recurrence and low median survival in many B-cell cancers highlight a need for new targeted therapeutic modalities. In dividing cells, eukaryotic translation initiation factor 5A (eIF5A) is hypusinated and involved in regulation of protein synthesis and proliferation, whereas the non-hypusinated form of eIF5A is a potent inducer of cell death in malignant cells. Here, we demonstrate the potential of modulating eIF5A expression as a novel approach to treating B-cell cancers. SNS01-T is a nonviral polyethylenimine-based nanoparticle, designed to induce apoptosis selectively in B-cell cancers by small interfering RNA–mediated suppression of hypusinated eIF5A and plasmid-based overexpression of a non-hypusinable eIF5A mutant. In this study, we show that SNS01-T is preferentially taken up by malignant B cells, inhibits tumor growth in multiple animal models of B-cell cancers without damaging normal tissues, and synergizes with the current therapies bortezomib and lenalidomide to inhibit tumor progression. The results collectively demonstrate the potential of SNS01-T as a novel therapeutic for treatment of a diverse range of B-cell malignancies.

Multi-nutrient supplement improves hormone ratio associated with cancer risk

BackgroundGynecological cancers are among the most common in women and are directly related to a variety of hormonal factors. One potential risk factor associated with developing a gynecological malignancy is the ratio of two hormone metabolites, 2-Hydroxyestrone (2-HE) and 16alpha-Hydroxyestrone (16alpha-HE). A number of botanical constituents such as indoles, flavonoids, and resveratrol have been shown to have a favorable effect on the metabolic pathways that affect this ratio. The present study was designed to evaluate if a multi-nutrient supplement containing targeted botanical constituents would affect the 2-HE/16 alpha-HE ratio in middle-aged women.MethodsA retrospective analysis was performed on 76 female patients (mean age 54 years) who received 2-HE/16 alpha-HE ratio assessments at two separate time points. The ratio assessment was part of standard care for women who presented with risk indicators associated with a high proliferative state. All patients who completed pre and post assessments were included. Sixty-five of the patients received a multi-nutrient supplement, Lucentia Peak®, during the study period. Eleven patients chose not to take the supplement, but did receive ratio assessments at similar time points as the treatment group, allowing for between group comparisons. Paired t-tests were used to compare the changes in the 2-HE and 16alpha-HE measures as well as their ratio, both within groups and between groups.ResultsThe results demonstrated a significant increase in the 2-HE/16alpha-HE ratio in the treated group (pre 0.38 to post 0.57, p<0.0001), and was significantly different (p=0.02) compared to the change in the control group (pre 0.65 to post 0.64). This change appears to be mediated primarily by an increase in the 2-HE level. Individually, 54 patients given Lucentia Peak® had increased ratios while 11 patients had a decrease. In the control group, 3 patients had an increase in their ratio and 8 patients had a decrease.ConclusionsThe results demonstrated that women receiving the Lucentia Peak® multi-nutrient supplement had significant increases in their 2-HE:16alpha-HE ratio, which appears to be mediated primarily by increasing the 2-HE levels. These results suggest further research on phytonutrients that might positively affect estrogen metabolism is warranted.

Prostate cancer immunology – an update for Urologists

A better understanding of the immune processes in the pathogenesis and progression of prostate cancer (CaP) may point the way towards improved treatment modalities. The challenge is to amplify immune responses to combat tumour escape mechanisms. Infection and inflammation may have a role in prostate carcinogenesis, including the newly discovered xenotropic murine leukaemia virus (XMRV). These inflammatory states damage defence mechanisms and induce a high proliferative state favouring further mutation and impaired immune surveillance. With this knowledge we are able to explore the use of immunotherapy to rejuvenate the immune system in combating CaP. Recently Sipuleucel-T, an immunotherapeutic agent for metastatic androgen independent CaP, has resulted in improved survival and might be the first immunotherapeutic agent to obtain approval for CaP treatment. This short review will focus on the growing body of evidence suggesting an immunity-based link between CaP and inflammation and infection.

Xenografts of Pediatric Acute Lymphoblastic Leukemia Retain the Gene Expression Pattern From the Diagnostic Material They Originated From Over Serial Passages.

Abstract 1629Poster Board I-655Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia. DisclosuresNo relevant conflicts of interest to declare.

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Activation of Mammalian Target of Rapamycin Signaling Promotes Cell Cycle Progression and Protects Cells from Apoptosis in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

328. Transcriptionally Targeted Cancer Gene Therapy for Small Cell Lung Cancer

Top of pageAbstract Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic disease for which there is currently no satisfactory treatment. Therefore development of novel treatment modalities is highly in demand. As SCLC is a disseminated disease, treatment must be administered systemically and therefore requires a high level of targeting. This can be achieved by transcriptionally targeted cancer gene therapy. We have identified promoters regions from several genes, which confer very high and SCLC specific mRNA expression - genes normally either only active during early developmental differentiation or associated with high proliferation. The insulinoma-associated 1 (INSM1) gene encodes a transcriptional repressor normally exclusively expressed transiently during early embryonic development in neuroendocrine regions, but has been found highly re-activated in neuroendocrine tumours, including SCLC. A 1.7 kb region of this promoter has very high activity in most SCLC cell lines and no activity in normal and other cancer cell lines. We have demonstrated that this promoter region confers sufficient activity when expressing a therapeutic gene to cause SCLC specific cell death in vitro and in vivo. The Achaete-Scute Homolog 1 (ASCL1) encoding a transcription factor transiently expressed during early development of neural and neuroendocrine progenitor cells, is also re-expressed in SCLC. A proximal 0.7 kb region of the ASCL1 promoter demonstrated high expression in a subset of SCLC cell lines with low or no expression in other cell lines. Addition of a suggested distal 2.5 kb repressor region gave no significant increase in specificity, but a general decrease in activity. The 0.7 kb promoter region regulating expression of a therapeutic gene was found to be sufficiently active to mediate SCLC specific cell death in vitro. The Enhancer of Zeste Homolog 2 (EZH2) gene is a member of the Polycomb group of proteins important for maintaining the silenced state of homeotic genes in the adult and is associated with high proliferation. EZH2 has been found highly overexpressed in a number of cancers, including SCLC. All tested cell lines (normal or cancer) have high expression of EZH2 mRNA, thus reflecting their high proliferative state. However, a 1.1 kb region of the EZH2 promoter displayed very high activity in SCLC cell lines, but not in other cell lines and was sufficiently active to mediate SCLC cell death in vitro. This short region of the EZH2 promoter has previously been shown to be activated by transcription factors from the E2F family. Preliminary results indicate that the high activity of the promoter region in SCLC is due to the lack of functional RB, which normally down regulates the activities of the E2F family. Therefore this short region of the promoter has potential for gene therapy for RB deficient cancers. The promoter regions from all three genes are currently being tested in mouse tumour models for activity and specificity using a novel liposomal delivery system for systemic application.

Clinical Significance of Apoptosis Is Independent from Proliferation in Acute Myeloid Leukemia (AML).

Multidrug resistance and recurrent disease are key problems in the variable response of AML pts to treatment. Tumor cells in a high proliferative state have a high density of transferrin receptors, as demonstrated in breast cancer (Yang DC, 2001) and in adult T-cell leukemia/lymphoma (Moura IC, 2004). On the other hand, defects in apoptotic pathways such as higher levels of bcl-2 and Mcl-1 were reported in “poor risk” AML pts. Current availability of antisense oligonucleotides targeted both to the transferrin receptor genes and bcl-2 incited us to evaluate the impact of proliferative and/or apoptotic pathways on AML prognosis. Therefore, a large series of 325 pts, affected by de novo AML, except FAB M3, median age 55 years, treated with intensive chemotherapy regimens, were studied. The aims of our research were: 1) to correlate bax/bcl-2 ratio with the proliferation levels, determined by the transferrin receptor (CD71) and 2) to demonstrate that the clinical significance of spontaneous apoptosis is independent from proliferation. CD71, bcl-2 and bax proteins were determined by multicolor flow cytometry. CD71 was evaluated as mean fluorescence intensity (MFI) and bax/bcl-2 ratio was obtained by dividing MFI bax/MFI bcl-2. One hundred-seventy five pts (53.8%) were bax/bcl-2 ratio positive and 204/324 (63%) were CD71 positive, respectively. There was a close correlation between higher CD71 expression and Ki-67 positive staining by flow cytometry (r=0.86), confirming that transferrin receptor overexpression is really linked to increased cellular proliferation in AML. No significant correlation was found between a higher bax/bcl-2 ratio and a lower CD71 MFI (p=0.16), confirming that an apoptosis resistant protein profile may have variable proliferation levels. A significant lower complete remission (CR) rate was found in pts with lower bax/bcl-2 ratio (43% vs 72%, p<0.00001) or higher CD71 (47% vs 74%, p=0.00001). Overall survival (OS) was significantly shorter either in pts with lower bax/bcl-2 ratio (1% vs 15% at 4 years; p=0.00001) or higher CD71 MFI (5% vs 104% at 5 years; p=0.002).Noteworthy, higher bax/bcl-2 ratio plus lower CD71 MFI identified an AML subset at better prognosis with regard to CR (91% vs 32%; p<0.00001) and OS (31% vs 0% at 2 years; p<0.00001). In order to confirm the independent prognostic value of bax/bcl-2 ratio from proliferation levels, we investigated the CD71 + (203 pts) and the CD71- (121 pts) AML subgroups. As a matter of fact, a lower CR rate was found in patients with lower bax/bcl-2 ratio either within CD71+ (34% vs 61%, p=0.0009) or CD71- (60% vs 90%, p=0.0006) pts. Also a lower bax/bcl-2 ratio was associated with a shorter OS both within CD71+ (2% vs 10% at 4 years, p=0.008) and within CD71- (0% vs 24% at 3.5 years, p=0.00008) pts. The independent prognostic value of bax/bcl-2 ratio was confirmed in multivariate analysis with regard to CR (p=0.00007) and OS (p=0.00003). The lack of significant correlation between bax/bcl-2 ratio and CD71 MFI confirms the independence of apoptosis from proliferation pathways. Furthermore, the capacity of bax/bcl-2 ratio of clearly identifying patients at different prognosis within the CD71+ and CD71- subgroups implies that apoptosis has an intrinsic more relevant clinical significance as compared to proliferation. That has to be taken in account when strategies are to be planned in order to resolve both chemoresistance and relapse in AML.

Expression of Mcl-1 in mantle cell lymphoma is associated with high-grade morphology, a high proliferative state, and p53 overexpression.

Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma characterized by the t(11;14)(q13;q32) and cyclin D1 overexpression. Defects in apoptosis may contribute to pathogenesis. This study evaluated the expression of the anti-apoptotic protein Mcl-1 in two MCL cell lines and five frozen MCL tumours (four small-cell, one blastoid/large-cell) using western blot analysis. Mcl-1 expression was also assessed in 36 formalin-fixed, paraffin wax-embedded MCL tumours (24 small-cell, 12 blastoid/large-cell) by immunohistochemistry. Western blot analysis revealed the expected 37 kD protein product in both MCL cell lines and in five frozen tumours, with the blastoid case having the highest expression level. Using a cut-off of >10% immunolabelled cells for Mcl-1, it was found that 12 of 36 MCL tumours were positive. Mcl-1-positive tumours had a higher frequency of blastoid/large-cell morphology (8/12 versus 4/24, p = 0.009), p53 overexpression (3/10 versus 1/23, p = 0.04), and higher Ki67 immuno-labelling (p = 0.002). It is concluded that expression of Mcl-1 in MCL is heterogeneous. A relatively high level of Mcl-1 expression correlates with high-grade morphology, a high proliferative state, and p53 overexpression.

Histopathological analysis of non-malignant and malignant epithelium in achalasia of the esophagus.

We studied the premalignant nature of achalasia using anti-Ki-67 and anti-p53 monoclonal antibodies immunohistochemically. In this study, four patients with esophageal carcinoma and achalasia were investigated. Three tumors were pT4 (UICC pTNM) and one tumor was pT1. The majority of non-malignant esophageal epithelium showed esophagitis and/or dysplasia histologically. Esophageal epithelial cells in the lesions of esophagitis and/or dysplasia had a higher number of Ki-67-positive cells than normal epithelial cells. p53 protein was expressed in two tumors and it was not expressed in non-malignant epithelium. From these results, we found that esophageal epithelium in achalasia lesions is changed to varying degrees of esophagitis and/or dysplasia by stagnation of intake foods, and these abnormal epithelial cells showed a high proliferative state compared with the normal cells without the p53 gene mutation. We suggest that the distinct proliferative status is a cause of carcinogenesis.

Expression of proliferating cell nuclear antigen in gastrointestinal tract lesions and its relationship to bcl-2 expression.

We report the relationship between proliferation as measured by the expression of proliferating cell nuclear antigen (PCNA) and the proto-oncogene bcl-2 in inflammatory and neoplastic lesions of the gastrointestinal tract and associated normal tissues. We found that (1) PCNA and bcl-2 are coexpressed in the stem cell-regenerative compartments of normal tissues but are not coexpressed in carcinomas or adjacent histologically normal epithelium, (2) there is marked heterogeneity of PCNA and bcl-2 expression in neoplastic lesions of the gastrointestinal tract, and (3) overexpression of PCNA is frequently seen in histologically inactive ulcerative colitis. We postulate (1) increased bcl-2 expression in epithelium adjacent to tumors represents an inherent change in the morphologically normal epithelium because it occurs without the corresponding high proliferative state seen in the normal crypt-regenerative compartment, (2) heterogeneity may provide a mechanistic explanation for chemotherapeutic resistance in tumors since cells having high bcl-2 but low proliferative activity would have prolonged survival and might show resistance to chemotherapeutic agents, and (3) the increased proliferative state in histologically inactive ulcerative colitis may provide a partial explanation for the increased risk of colon carcinomas in these patients.

Role of Persistent, Non-Genotoxic Tissue Damage in Rodent Cancer and Relevance to Humans

This review has focused on the importance of cellular proliferation in neoplasia induced by nongenotoxic carcinogens and has not attempted to address the mechanisms involved in the progression from the hyperplastic to the neoplastic state. Major advances in this area are likely in the next decade and should explain how cells in an abnormally high proliferative state are rendered more vulnerable either to the action of endogenous or environmental mutagens or to defects in cell reproduction, thus providing the stimulus for progression to neoplasia (165). In this review evidence has been presented to support the hypothesis that sustained tissue damage induced by nongenotoxic compounds in rodents predisposes to tumor development. The evidence has been obtained by drawing from the published findings of experimental rodent studies conducted over the past 50 years. The experience gained at various sites in the rodent including the connective tissue, liver, bladder, and forestomach shows the existence of a threshold dose for various test materials/agents, below which neither sustained tissue damage nor tumor induction occurs but above which level both effects are manifest. Taken collectively, an overall picture emerges that sustained cell proliferation renders various sites in the rodent vulnerable to tumor development. The validity of this hypothesis is of importance to the evaluation of carcinogenic risk of chemicals to humans. For those nongenotoxic carcinogens known to cause characteristically defined and sustainable tissue damage as a precursor of tumor development in rodents, it should be possible to establish a threshold dose below which both effects disappear and upon which a safety margin in humans can be based. Some limited evidence supports an association between chronic tissue injury and neoplastic development in humans. Note, however, that certain types of induced tissue damage may be rodent-specific and therefore have no relevance for humans. We conclude that the appearance of persistent tissue damage predisposes to tumor development in rodents exposed to nongenotoxic carcinogens and that systematic studies in rodents should provide a rational basis for arriving at a safety margin in humans exposed to such agents.