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Background : DNA-dependent protein kinase (DNA-PK) is a validated cancer therapeutic target involved in DNA damage response (DDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). Ku serves as a sensor of DSBs by binding to DNA ends and activating DNA-PK. Inhibition of DNA-PK is a common strategy to block DSB repair and improve efficacy of ionizing radiation (IR) therapy and radiomimetic drug therapies. We have previously developed Ku-DNA binding inhibitors (Ku-DBis) that block in vitro and cellular NHEJ activity, abrogate DNA-PK autophosphorylation, and potentiate cellular sensitivity to IR. Results and Conclusions : Here we report the discovery of oxindole Ku-DBis with improved cellular uptake and retained potent Ku-inhibitory activity. Variable monotherapy activity was observed in a panel of non-small cell lung cancer (NSCLC) cell lines, with ATM-null cells being the most sensitive and showing synergy with IR. BRCA1-deficient cells were resistant to single-agent treatment and antagonistic when combined with DSB-generating therapies. In vivo studies in an NSCLC xenograft model demonstrated that the Ku-DBi treatment blocked IR-dependent DNA-PKcs autophosphorylation, modulated DDR, and reduced tumor cell proliferation. This represents the first in vivo demonstration of a Ku-targeted DNA-binding inhibitor impacting IR response and highlights the potential therapeutic utility of Ku-DBis for cancer treatment.

Clonal mutations represent the initiating molecular defects related to cellular transition of a normal phenotype to a malignant phenotype. Molecular genomic assessment utilizing next generation and whole exome sequencing is now being increasingly applied to biomarker determination to refine the use of targeted immune therapies. Case examples followed by retrospective study assessment have convincingly demonstrated clonal neoantigens provide a relevant predictor of response to checkpoint inhibition. A meta-analysis, by Litchfield et al., of over 1000 cancer patients from 12 landmark trials demonstrated no clinical benefit to checkpoint inhibitor (CPI) therapy in correlation to high subclonal tumor mutational burden (TMB), whereas high clonal TMB was found to be significantly correlated with better overall survival ( p = 0.000000029). We discuss the mechanism of clonal vs. subclonal neoantigen targeting relationship to homologous recombination proficient (HRP) profile, evidence of preclinical and clinical benefit related to clonal neoantigens, and review a novel developing therapy called Vigil ® , designed to expand the clonal neoantigen targeting effector cell populations. Vigil ® is an autologous cellular immunotherapy which is designed to carry the full set of personal clonal neoantigens. Phase 2b results demonstrate a durable recurrence-free survival (RFS) and overall survival (OS) advantage for Vigil ® in a subset ovarian cancer population with an HRP cancer profile.

An increasing number of human diseases, including allergies, infections, inflammation, and cancer, involve roles for basophils. Traditionally viewed as the rarest leukocytes that are present only in the circulation, basophils have recently emerged as important players in systemic as well as tissue-specific immune responses. Their functions are regulated by immunoglobulins (Igs), and this enables basophils to integrate diverse adaptive and innate immunity signals. IgE is well known to regulate basophil responses in the context of type 2 immunity and allergic inflammation; however, growing evidence shows that IgG, IgA, and IgD also shape specific aspects of basophil functions relevant to many human diseases. We discuss recent mechanistic advances underpinning antibody-mediated basophil responses and propose strategies for the treatment of basophil-associated disorders., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In-depth insights in cancer biology over the past decades have highlighted the important roles of epigenetic mechanisms in the initiation and progression of tumorigenesis. The cancer epigenome usually experiences multiple alternations, including genome-wide DNA hypomethylation and site-specific DNA hypermethylation, various histone posttranslational modifications, and dysregulation of non-coding RNAs (ncRNAs). These epigenetic changes are plastic and reversible, and could potentially occur in the early stage of carcinogenesis preceding genetic mutation, offering unique opportunities for intervention therapies. Therefore, targeting the cancer epigenome or cancer epigenetic dysregulation with some selected agents (called epi-drugs) represents an evolving and promising strategy for cancer chemoprevention and therapy. Phytochemicals, as a class of pleiotropic molecules, have manifested great potential in modulating different cancer processes through epigenetic machinery, of which green tea polyphenol epigallocatechin-3-gallate (EGCG) is one of the most extensively studied. In this review, we first summarize epigenetic events involved in the pathogenesis of cancer, including DNA/RNA methylations, histone modifications and ncRNAs' dysregulations. We then focus on the recently discovered roles of phytochemicals, with a special emphasis on EGCG, in modulating different cancer processes through regulating epigenetic machinery. We finally discuss limitations of EGCG as an epigenetic modulator for cancer chemoprevention and treatment and offer potential strategies to overcome the shortcomings., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Metformin is being actively repurposed for the treatment of gynecologic malignancies including ovarian cancer. We investigated if metformin induces analogous metabolic changes across ovarian cancer cells. Functional metabolic analysis showed metformin caused an immediate and sustained decrease in oxygen consumption while increasing glycolysis across A2780, C200, and SKOV3ip cell lines. Untargeted metabolomics showed metformin to have differential effects on glycolysis and TCA cycle metabolites, while consistent increased fatty acid oxidation intermediates were observed across the three cell lines. Metabolite set enrichment analysis showed alpha-linolenic/linoleic acid metabolism as being most upregulated. Downstream mediators of the alpha-linolenic/linoleic acid metabolism, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were abundant in all three cell lines. EPA was more effective in inhibiting SKOV3 and CaOV3 xenografts, which correlated with inhibition of inflammatory markers and indicated a role for EPA-derived specialized pro-resolving mediators such as Resolvin E1. Thus, modulation of the metabolism of omega-3 fatty acids and their anti-inflammatory signaling molecules appears to be one of the common mechanisms of metformin's antitumor activity. The distinct metabolic signature of the tumors may indicate metformin response and aid the preclinical and clinical interpretation of metformin therapy in ovarian and other cancers.

Benzo[a]pyrene (BaP) can cause hepatorenal toxicity. Secoisolariciresinol diglucoside (SDG), a polyphenolic compound present in flaxseed, has shown a variety of biological activities including antioxidant, anti-inflammatory, anti-apoptotic effects. This study aimed to investigate the protective effects and working mechanisms of SDG against BaP-induced hepatorenal injury. Forty male mice were administrated daily (via gastric gavage; 4 weeks) with 0.9% saline (control), BaP (75 mg/kg body weight (b.w.)), SDG (100 mg/kg b.w.), SDG (100 mg/kg b.w.)+BaP (75 mg/kg b.w.). Results showed that the mice treated with SDG + BaP had significantly (P < 0.05) higher body weight, lower organ-to-body weight ratio, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) activities, and less levels of serum creatinine (CRE) and blood urea nitrogen (BUN) than those treated with BaP alone. SDG administration alleviated BaP-induced oxidative damages, inflammation and apoptosis. Furthermore, it significantly (P < 0.05) downregulated phosphor-p38 (p-p38) and phosphor-extracellular regulated protein kinases (p-ERK) levels, upregulated mitogen-activated protein kinase phosphatase-1 (MKP-1) level, and suppressed miR-101a expression compared with BaP alone group. Taken together, these results showed for the first time that SDG has protective effects against BaP-induced liver and kidney toxicity in mice through regulating oxidative stress, inflammation and apoptosis via miR-101a/MKP-1-mediated p38 and ERK pathway., (Copyright © 2021. Published by Elsevier Ltd.)

Lung cancer is one of the most common cancers in the world. Although medical treatment has made impressive progress in recent years, it is still one of the leading causes of cancer-related deaths in men and women. Ferroptosis is a type of non-apoptotic cell death modality, usually characterized by iron-dependent lipid peroxidation, rather than caspase-induced protein cleavage. Excessive or lack of ferroptosis is associated with a variety of diseases, including cancer and ischaemia-reperfusion injury. Recent preclinical evidence suggests that targeting ferroptotic pathway is a potential strategy for the treatment of lung cancer. In this review, we summarize the core mechanism and regulatory network of ferroptosis in lung cancer cells, and highlight ferroptosis induction-related tumor therapies. The reviewed information may provide new insights for targeted lung cancer therapy., (© 2021. The Author(s).)

Background: Breast cancer (BC) incidence and mortality are lower in Poland than in the United States (US). However, Polish-born migrant women to US approach the higher BC mortality rates of US women. We evaluated the association between consumption of cabbage/sauerkraut foods and BC risk in Polish-born migrants to US., Methods: We conducted a case-control study of BC among Polish-born migrants in Cook County and the Detroit Metropolitan Area. Cases ( n = 131) were 20-79 years old with histological/cytological confirmation of invasive BC. Population-based controls ( n = 284) were frequency matched to cases on age and residence. Food frequency questionnaires assessed diet during adulthood and age 12-13 years. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated with conditional logistic regression. Consumption of total, raw/short-cooked, and long-cooked cabbage/sauerkraut foods was categorized as low, medium, or high (frequency of servings/week)., Results: Higher consumption of total and raw/short-cooked cabbage/sauerkraut foods, during both adolescence and adulthood, was associated with a significantly lower BC risk. Consumption of long-cooked cabbage/sauerkraut foods was low and not significantly associated with risk. The multivariate OR for total cabbage/sauerkraut consumption, high vs. low (>4 vs. ≤2 servings/week) during adolescence was 0.36 (95% CI = 0.18-0.71, p trend < 0.01) and 0.50 (95% CI = 0.23-1.06, p trend = 0.08) during adulthood. For raw/short-cooked cabbage/sauerkraut (>3 vs. ≤1.5 servings/week), the ORs were 0.35 (95% CI = 0.16-0.72, p trend < 0.01) during adolescence and 0.37 (95% CI = 0.17-0.78, p trend < 0.01) during adulthood. For joint adolescent/adult consumption of raw/short-cooked cabbage/sauerkraut foods, (high, high) vs. (low, low), the OR was 0.23 (95% CI = 0.07-0.65). The significant association for high adolescent consumption of raw/short-cooked cabbage/sauerkraut foods and reduced BC risk was consistent across all levels of consumption in adulthood., Conclusion: Greater consumption of total and raw/short-cooked cabbage/sauerkraut foods either during adolescence or adulthood was associated with significantly reduced BC risk among Polish migrant women. These findings contribute to the growing literature suggesting a protective effect of a potentially modifiable factor, cruciferous vegetable intake, on breast cancer risk.

The development of targeted therapies that inhibit cancer-driving oncogenes has improved outcomes of patients diagnosed with lung adenocarcinoma (LUAD). In contrast, patients diagnosed with lung squamous cell carcinoma (LUSC) suffer worse survival outcomes and lack effective targeted treatment options. Identification of molecular drivers of LUSC to support development of targeted treatments is urgently needed. Addressing this need, the current report introduces the novel cancer gene SLIT- and NTRK-like family member 3 (SLITRK3) and its role in activating the neurotrophic receptor tyrosine kinase 3 (NTRK3) in LUSC cells. Multiple genome-wide data sets from patient samples were produced by us or downloaded from public databases to analyze tumor gene copy number aberrations, mRNA expression and associated survival outcomes. An accompanying mechanistic study employed LUSC cell lines and multiple methods, including in situ immunofluorescence, sphere-formation assay, and fluorescence-activated cell sorting analysis of the CD133-positive cell fraction. Altogether, the results indicate that gene amplification and consequent high expression of SLITRK3 in LUSC is associated with worse outcomes and induces SLITRK3-dependent activation of NTRK3 to promote a cancer stem cell phenotype that is inhibited by existing NTRK-targeted inhibitors. Based on a recent literature search, this is the first report of a mechanistic role for SLITRK3 in cancer., (© 2021. The Author(s).)

Limited information is available on the impact of intensity of conditioning regimens in haploidentical peripheral blood stem cell transplant (haploPBSCT) with post-transplant cyclophosphamide (PTcy). We retrospectively compared outcomes of haplo-PBSCT between myeloablative (MAC) (n = 24) and reduced intensity conditioning (RIC) regimens (n = 65). Propensity score-based multivariable analyses were performed to adjust confounding effects of baseline characteristics between both groups. Eighty-nine patients underwent haplo-PBSCT between January 2012 and June 2019. For MAC and RIC, the cumulative incidences of grade III--IV acute GVHD were 4.2% and 3.1%, respectively (p = 0.92), and chronic GVHD were 18.9% and 36.5%, respectively (p = 0.08). Median follow-up for overall survival (OS) after MAC and RIC was 1.86 and 2.2 years, respectively. For MAC and RIC, one-year OS was 68.8% and 67.4%, respectively (p = 0.85); one-year relapse rate was 22.4% and 18.3%, respectively (p = 0.74); one-year relapse-free survival (RFS) was 56% and 59.7%, respectively (p = 0.87); and one-year non-relapse mortality (NRM) was 22% and 21.9%, respectively (p = 0.58). Using propensity score-based multivariable analyses, no difference in OS (HR 0.72, p = 0.51), relapse (SHR 0.63, p = 0.42), RFS (HR 0.74, p = 0.49) and NRM (SHR 1.11, p = 0.87) was noted between RIC and MAC. Our study shows no difference in outcomes between MAC and RIC regimens in haplo-PBSCT.

The ubiquitin-proteasome system (UPS) plays a central role in the degradation of cellular proteins. Targeting protein degradation has been validated as an effective strategy for cancer therapy since 2003. Several components of the UPS have been validated as potential anticancer targets, including 20S proteasomes, 19S proteasome-associated deubiquitinases (DUBs) and ubiquitin ligases (E3s). 20S proteasome inhibitors (such as bortezomib/BTZ and carfilzomib/CFZ) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) and some other liquid tumors. Although survival of MM patients has been improved by the introduction of BTZ-based therapies, these clinical 20S proteasome inhibitors have several limitations, including emergence of resistance in MM patients, neuro-toxicities, and little efficacy in solid tumors. One of strategies to improve the current status of cancer treatment is to repurpose old drugs with UPS-inhibitory properties as new anticancer agents. Old drug reposition represents an attractive drug discovery approach compared to the traditional de novo drug discovery process which is time-consuming and costly. In this review, we summarize status of repurposed inhibitors of various UPS components, including 20S proteasomes, 19S-associated DUBs, and ubiquitin ligase E3s. The original and new mechanisms of action, molecular targets, and potential anticancer activities of these repurposed UPS inhibitors are reviewed, and their new uses including combinational therapies for cancer treatment are discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Yin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

The ubiquitin proteasome system has been validated as a target of cancer therapies evident by the US FDA approval of anticancer 20S proteasome inhibitors. Deubiquitinating enzymes (DUBs), an essential component of the ubiquitin proteasome system, regulate cellular processes through the removal of ubiquitin from ubiquitinated-tagged proteins. The deubiquitination process has been linked with cancer and other pathologies. As such, the study of proteasomal DUBs and their inhibitors has garnered interest as a novel strategy to improve current cancer therapies, especially for cancers resistant to 20S proteasome inhibitors. This article reviews proteasomal DUB inhibitors in the context of: discovery through rational design approach, discovery from searching natural products and discovery from repurposing old drugs, and offers a future perspective.

Resistance to targeted therapeutics is a key issue limiting the long-term utility of these medications in the management of molecularly selected subsets of cancer patients, including patients with non-small cell lung cancer harboring oncogenic alterations affecting EGFR , ALK and other genes. Bypass resistance mediated by activation of MET kinase has emerged as a frequent, validated and pivotal resistance mechanism in multiple types of cancers. Biochemical understanding is accumulating to explain the unique role of MET in such bypass pathways, providing alternate downstream activation opportunities and intricate interactions during epithelial-mesenchymal transitions. Multiple diagnostic testing platforms have become available for selecting appropriate patients for MET targeting in a variety of settings. Importantly, in light of the failures of several earlier clinical studies of MET targeting agents, a large array of recent and current MET-focused trials are incorporating stricter patient selection and more robust predictive biomarkers providing hope for validation of MET targeting as a clinically impactful strategy., Competing Interests: The authors have no conflicts of interest to declare.

Photochemical control over irreversible inhibition was shown using Ru(ii)-caged inhibitors of cathepsin L. Levels of control were dependent on where the Ru(ii) complex was attached to the organic inhibitor, reaching >10 : 1 with optimal placement. A new strategy for photoreleasing Ru(ii) fragments from inhibitor-enzyme conjugates is also reported.

Gene amplification is a common mechanism of oncogene activation in cancer. Several large-scale efforts aimed at identifying the comprehensive set of genomic regions that are recurrently amplified in cancer have been completed. In breast cancer, these studies have identified recurrently amplified regions containing known drivers such as HER2 and CCND1 as well as regions where the driver oncogene is unknown. In this study, we integrated RNAi-based functional genetic data with copy number and expression data to identify genes that are recurrently amplified, overexpressed and also necessary for the growth/survival of breast cancer cells. Further analysis using clinical data from The Cancer Genome Atlas specifically identified candidate genes that play a role in determining patient outcomes. Using this approach, we identified two genes, TCP1 and CCT2, as being recurrently altered in breast cancer, necessary for growth/survival of breast cancer cells in vitro, and determinants of overall survival in breast cancer patients. We also show that expression of TCP1 is regulated by driver oncogene activation of PI3K signaling in breast cancer. Interestingly, the TCP1 and CCT2 genes both encode for components of a multi-protein chaperone complex in the cell known as the TCP1 Containing Ring Complex (TRiC). Our results demonstrate a role for the TRiC subunits TCP1 and CCT2, and potentially the entire TRiC complex, in breast cancer and provide rationale for TRiC as a novel therapeutic target in breast cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Our laboratory has had a longstanding interest in how the interactions between tumors and their microenvironment affect malignant progression. Recently, we have focused on defining the proteolytic pathways that function in the transition of breast cancer from the pre-invasive lesions of ductal carcinoma in situ (DCIS) to invasive ductal carcinomas (IDCs). We use live-cell imaging to visualize, localize and quantify proteolysis as it occurs in real-time and thereby have established roles for lysosomal cysteine proteases both pericellularly and intracellularly in tumor proteolysis. To facilitate these studies, we have developed and optimized 3D organotypic co-culture models that recapitulate the in vivo interactions of mammary epithelial cells or tumor cells with stromal and inflammatory cells. Here we will discuss the background that led to our present studies as well as the techniques and models that we employ. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.25 ml of plasma with 10 microl of 1 M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C(18) column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1-2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration-time profile of ABT-888 after its oral administration in cancer patients., (2009 Elsevier B.V. All rights reserved.)

The ability of tumor cells to adhere to, migrate on, and remodel extracellular matrices is mediated by cell surface receptors such as beta1-integrins. Here we conducted functional live-cell imaging in real time to investigate the effects of modulating beta1-integrin expression and function on proteolytic remodeling of the extracellular matrix. Human breast and prostate cancer cells were grown on reconstituted basement membrane containing a quenched fluorescent form of collagen IV. Generation of cleavage products and the resulting increases in fluorescence were imaged and quantified. Decreases in the expression and activity of beta1-integrin reduced digestion of quenched fluorescent-collagen IV by the breast and prostate cancer cells and correspondingly their invasion through and migration on reconstituted basement membrane. Decreased extracellular matrix degradation also was associated with changes in the constituents of proteolytic pathways: decreases in secretion of the cysteine protease cathepsin B, the matrix metalloproteinase (MMP)-13, and tissue inhibitors of metalloproteinases (TIMP)-1 and 2; a decrease in expression of MMP-14 or membrane type 1 MMP; and an increase in secretion of TIMP-3. This is the first study to demonstrate through functional live-cell imaging that downregulation of beta1-integrin expression and function reduces proteolysis of collagen IV by breast and prostate cancer cells.

This study was performed to determine the dose limiting toxicity (DLT), the recommended phase II dose and the pharmacokinetic profile for SR271425, given over 1 h every 3 weeks. The initial starting dose of SR271425 was 17 mg/m(2). Patient selection was based on common phase I criteria as well as additional cardiac criteria. Thirty-eight patients were accrued to 16 dose levels from 17 to 1,320 mg/m(2). Patient characteristics included 24 males and 14 females ages 35-78 with an Eastern Cooperative Oncology Group performance status of 0 (ten patients), 1 (27) and 2 (1). Tumor types were typical for a phase I study. The maximum administered dose was 1,320 mg/m(2) with two DLTs, both QTc grade 3 prolongation. No drug related hematological toxicity was noted. Grade 1 toxicities included rash, flushing, pruritus, weight loss, diarrhea, hypertension and fatigue. Grade 2 toxicities included yellow discoloration of the skin, nausea and vomiting. QTc prolongation and hyperbilirubinemia were the only grade 3 toxicities noted. No confirmed tumor response was observed; however, two patients had prolonged stable disease. Both C(end) and area under the plasma concentration-time curve increased in a dose related manner. Plasma drug concentrations declined in a biphasic manner with a mean terminal elimination half-life (t (1/2)) of 7.1 h (+/-1.3). There was no change in clearance or volume of distribution over the dose range studied. Due to cardiac toxicity occurring with both the parent compound, SR233377, as well as this analog, this series of agents was abandoned from further clinical development.

The process of cancer progression involves the action of multiple proteolytic systems, among which the family of matrix metalloproteinases (MMPs) play a pivotal role. The MMPs evolved to accomplish their proteolytic tasks in multiple cellular and tissue microenvironments including lipid rafts by incorporation and deletions of specific structural domains. The membrane type-MMPs (MT-MMPs) incorporated membrane anchoring domains that display these proteases at the cell surface, and thus they are optimal pericellular proteolytic machines. Two members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP), are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor, which confers these enzymes a unique set of regulatory and functional mechanisms that separates them from the rest of the MMP family. Discovered almost a decade ago, the body of work on GPI-MT-MMPs today is still surprisingly limited when compared to other MT-MMPs. However, new evidence shows that the GPI-MT-MMPs are highly expressed in human cancer, where they are associated with progression. Accumulating biochemical and functional evidence also highlights their distinct properties. In this review, we summarize the structural, biochemical, and biological properties of GPI-MT-MMPs and present an overview of their expression and role in cancer. We further discuss the potential implications of GPI-anchoring for enzyme function. Finally, we comment on the new scientific challenges that lie ahead to better understand the function and role in cancer of these intriguing but yet unique MMPs.

Dietary intake of lycopene and soy has been associated with a lower risk of prostate cancer. In vitro studies with lycopene and genistein, a soy isoflavone, have shown induction of apoptosis and inhibition of cell growth in androgen-sensitive (LNCaP) and androgen-independent (PC3 and VeCaP) prostate cancer cell lines. In a previous Phase II clinical trial in prostate cancer patients, we observed prostate-specific antigen (PSA) stabilization with soy isoflavone intake. In this Phase II clinical trial, we investigated the efficacy of lycopene alone or in combination with soy isoflavones on serum PSA levels in men with prostate cancer. To be eligible for the study, men with prostate cancer had to have rising serum PSA following local therapy or while on hormone therapy. Study population included 71 eligible patients who had 3 successive rising PSA levels or a minimum PSA of 10 ng/ml at 2 successive evaluations prior to starting therapy. Subjects were randomly assigned to receive a tomato extract capsule containing 15 mg of lycopene alone (n = 38) or together with a capsule containing 40 mg of a soy isoflavone mixture (n = 33) twice daily orally for a maximum of 6 mo. One patient on the lycopene arm did not receive therapy due to his inability to ingest the study pill. There was no decline in serum PSA in either group qualifying for a partial or complete response. However, 35 of 37 (95%) evaluable patients in the lycopene group and 22 of 33 (67%) evaluable patients in the lycopene plus soy isoflavone group achieved stable disease described as stabilization in serum PSA level. The data suggest that lycopene and soy isoflavones have activity in prostate cancer patients with PSA relapse disease and may delay progression of both hormone-refractory and hormone-sensitive prostate cancer. However, there may not be an additive effect between the 2 compounds when taken together. Future studies are warranted to further investigate the efficacy of lycopene and soy isoflavones in prostate cancer as well as the mechanism of potential negative interaction between them.

The outcome for children with acute megakaryoblastic leukemia (AMKL) remains poor, except for cases associated with Down syndrome (DS). This study compared immunophenotypic and drug sensitivity patterns of childhood AMKL cases with or without DS. All DS-AMKL cases showed high expression of CD36 and were very sensitive to cytarabine and daunorubicin in vitro. In children without DS, high expression of CD36 on AMKL blasts identified a sub-group with immunophenotypic and drug sensitivity patterns similar to that of DS AMKL. Among non-DS AMKL patients, CD36-high cases had a superior outcome compared with CD36-negative cases. CD36 expression in acute myeloid leukemia cases other than AMKL was not associated with increased in vitro drug sensitivity. CD36 expression in AMKL may be an indicator of megakaryoblast maturation and chemotherapy sensitivity.

Atm is a stress-induced DNA damage checkpoint protein kinase with multiple roles in cell-cycle progression. Recent evidence indicates that Atm also plays a role in stem cell maintenance and self-renewal. It is not known whether Atm has a role during tissue regeneration. Using liver regeneration as a model system, we examined the role of Atm in this process. Here, we show that the expression levels of Atm protein were gradually increased during liver regeneration and this was correlated with the onset of DNA replication. The induction of Stat3 and JNK signaling, which are essential processes in normal regeneration response, was attenuated during the early phases of liver regeneration in Atm-deficient mice. P53 was transiently phosphorylated at serine 23 during liver regeneration in an Atm-dependent manner. In addition, we found that cyclin A induction was delayed and p21 was over-expressed, both of these processes were correlated with reduced and delayed DNA replication in Atm(-/-) mice during liver regeneration. Finally, we show that increased apoptosis was observed in Atm(-/-) mice in response to partial hepatectomy, indicating that Atm is required for the survival of hepatocytes. Collectively, these data indicate that liver regeneration is impaired in Atm-deficient mice. Given that liver is the first line of defense against environmental toxins, the elucidation of the function of Atm and Atm-mediated signaling pathways in liver metabolism and in response to environmental toxins is of fundamental interest.

Risk-based treatment strategies have improved outcome in childhood B-precursor acute lymphoblastic leukemia, and in vitro drug sensitivity assessment using methyl-thiazol-tetrazolium (MTT) assay has been shown to be an independent prognostic marker. To date, such strategies in childhood T-cell acute lymphoblastic leukemia (T-ALL) have proved elusive, and in vitro drug sensitivity testing has had limited success in T-ALL due to poor T-cell lymphoblast survival in vitro. We have developed the flow cytometric drug sensitivity assay (FCDSA) to evaluate in vitro drug sensitivity. We studied 68 cases of childhood T-ALL for cytarabine (Ara-C) and daunorubicin sensitivity by FCDSA and compared the results with those obtained by MTT assay. Spontaneous apoptosis was correlated with cytotoxicity rates for both drugs by FCDSA, but not with the results obtained by MTT assay. Daunorubicin sensitivity had a positive correlation with Ara-C in individual cases by FCDSA; but not by MTT assay. Studies repeated on stored samples had comparable results for both drugs by FCDSA (P<0.01), but not for Ara-C by MTT assay. Comparison of T-ALL sensitivity with acute myeloid leukemia (AML) cases revealed a unique pattern difference. Median cytotoxicity (expressed in arbitrary units) for Ara-C was 8 (0-47) and 27 (0-81), and daunorubicine cytotoxicity for T-ALL and AML samples was 79 (5-100) and 34 (0-98), respectively. Although age or white blood cell count at diagnosis was not associated with any particular drug response pattern, CD13 expression on T-lymphoblasts was associated with in vitro resistance. FCDSA is a reliable, practical and reproducible method that can be integrated into studies of drug-target cell interactions in T-ALL.

Loss of heterozygosity (LOH) is the most consistent genetic change in prostate cancer (CaP). We aimed, to correlate specific LOH and the overall LOH frequency, to disease progression after radical prostatectomy (RP) in high-grade CaP. Between January 1990 through December 1998, 126 patients who underwent RP (cT1-T2), Gleason 8-10, were pT3, or pN1, or SM(+) (surgical margins). Nine were lost of follow-up, 39/117 (33%) had no biochemical progression (mean follow-up: 45 months). After exclusion for preoperative PSA >50 ng/mL, a case-control study was designed by matching 26 of these cases with 26 similar patients without biochemical progression (criteria: pT, pN, year of surgery). Using microsatellite markers, LOH were assessed on six chromosomal regions (7q31, 8p22, 12p13, 13q14, 16q23.2, 18q21). No prognostic value was associated with LOH at any one specific locus. However, the overall LOH frequency (five classes, cutoff of 60%), was significantly higher if progression (P = 0.02; P = 0.03) in SM(+) patients, and was near statistical significance (P = 0.08; P = 0.07) for the overall case-control population. In multivariate analysis (overall population), the overall LOH rate > or =60% was independently associated with progression [P = 0.035; Odds Ratio (OR) = 5.54]. An overall LOH rate > or =60% predicted poor outcome in 85% of SM(+) patients and 69% of the whole population. Our results suggest that the overall rate of LOH at chromosomal "hot spots" is more likely to be predictive of recurrence than the presence of LOH at any one particular locus. Moreover, the identification of a threshold of LOH could help in predicting patients with poor outcome who may be candidates for local or systemic adjuvant therapies.

We retrospectively analyzed the prognostic significance of mixed chimerism and associated clinical parameters in 80 patients following unmanipulated allogenic stem cell transplantation. Chimerism studies were performed on marrow aspirates using fluorescent in situ hybridization and variable number tandem repeats techniques at day +30, day +90 and +12 months. The median overall survival (OS) was 24 months (range, 1-56 months). Mixed chimerism was found in 23, 28 and 14% of patients at day +30 (1 month), +90 (3 months), and +12 months, respectively. Day +30 chimerism studies failed to provide any prognostic information. Day +90 mixed chimeras (MC) had significantly higher relapse rates compared to day +90 complete chimeras (CC) at 6 months (P=0.03) and 18 months when compared to MC (P=0.03) following transplant. The median OS in day +90 MC and day+90 CC were, respectively (95% CI, 2-35 months), compared to 47 months (95% CI, 20-74 months) (P=0.02). In conclusion, chimerism studies on day +30 could be reserved for patients who fail to demonstrate engraftment. Day +90 MC had higher relapse rates and lower OS, and therefore may be considered for novel therapies and future studies.

Aims: Because of the observation of an abundance of leukaemia/lymphoma cell microparticles in the bone marrow aspiration sample of a patient with Burkitt's leukaemia at diagnosis, the occurrence of this phenomenon in leukaemia/lymphoma samples with available immune phenotyping data was investigated retrospectively., Methods: Flow cytometric immune phenotyping and spontaneous apoptosis analysis of the bone marrow mononuclear cell preparation of the index case were performed. Microparticles isolated form the bone marrow sample were also studied for the presence of leukaemia/lymphoma cell microparticles. List mode analysis of 225 cases of acute leukaemia or lymphoma with previously performed immune phenotyping was also carried out., Results: The presence of leukaemia/lymphoma cell microparticles could be detected by flow cytometry and they were found to be different from apoptotic bodies. Leukaemia/lymphoma cell microparticles were released in all cases of mature B cell neoplasms studied, although this phenomenon was rare in precursor B cell disorders and acute myeloid leukaemia., Conclusions: The generation of leukaemia/lymphoma cell microparticles in mature B cell neoplasms appears to be a common phenomenon. The pathogenesis and clinical implications must be investigated.

Oxidative DNA damage in blood appears to be useful as a marker of systemic oxidative stress levels. Dietary factors such as fat and energy intakes have been indicated to affect oxidative stress levels, and this may be an important mechanism by which diet can modulate cancer risk. The primary objective of this study was to investigate the effects of dietary intervention in premenopausal women on the levels of one type of oxidative DNA damage: 5-hydroxymethyl-2'-deoxyuridine. The trial randomly assigned women to control, low-fat, low-energy or combination low-fat/low-energy diets for 12 weeks. Blood samples were obtained every 2 weeks, and DNA was analysed for the levels of 5-hydroxymethyl-2'-deoxyuridine. Levels of DNA damage declined with time in each diet arm, including the control arm. The decreases were greater in the two arms with low-energy intake, but not significantly so. The numbers of women who exhibited decreased 5-hydroxymethyl-2'-deoxyuridine levels at 12 weeks versus baseline levels, however, was significantly greater in women assigned to any intervention diet (79%) than in the control arm (50%). Low-fat and low-energy diets therefore had a small effect on changes in oxidative DNA damage levels. The women participating in this study were not selected on the basis of increased cancer risk; therefore, they may have had low baseline levels of damage that were not amenable to further reduction by dietary change.

Purpose: To compare the pharmacokinetics and tissue distribution (both normal and tumor) of cryptophycin 52 (C-52) and its putative chlorohydrin prodrug cryptophycin 55 (C-55) in a murine model and to investigate a possible mechanism behind the superior activity of C-55., Methods: Mammary adenocarcinoma 16/c tumor-bearing mice were treated with an i.v. bolus of 11 mg/kg C-52 or 38 mg/kg C-55 in Cremophor-alcohol. At predetermined time intervals, C-52 and C-55 concentrations in plasma, liver, kidney, small intestine and tumors were measured using a previously described HPLC method. Pharmacokinetic parameters were computed using noncompartmental methods. Tissue (both normal and tumor) to plasma ratios as a function of time were also calculated for comparison., Results: Both C-52 and C-55 were rapidly distributed into different tissues including tumors following i.v. administration. However, the affinities of these compounds towards different tissues were different. Thus, the half-lives (minutes) of C-55 were in the decreasing order liver (725), intestine (494), tumor (206), kidney (62) and plasma (44), whereas the AUC values (microg x min/ml) were in the order tumor (9077), liver (7734), kidney (6790), plasma (2372) and intestine (2234). For C-52, the half-lives (minutes) were in the decreasing order liver (1333), kidney (718), intestine (389), tumor (181) and plasma (35), and the AUC values (microg x min/ml) were in the order kidney (1164), liver (609), intestine (487), plasma (457) and tumor (442). The relative exposures to C-52 after i.v. injection of C-55 were plasma 3.9%, tumor 80.8%, kidney 3.4%, liver 1.1% and intestine 2.8%. Although plasma exposure to C-52 following C-55 administration was relatively small, the use of C-55 to deliver C-52 increased the retention of C-52 and its AUC in tumor compared to direct injection of C-52. Simultaneously, this approach shortened C-52 retention in all normal tissues studied., Conclusions: The distribution of C-55 and its bioconversion to C-52 in different organs and tumor tissue observed in this study suggest the ability of C-55 to target tumor tissue, creating a depot of C-52 in tumor. Increased C-52 exposure of tumor, with concomitant decreased exposure of normal tissue, is a contributing factor to the superior activity of C-55 versus C-52. However, except in the case of tumor tissue in which 81% of C-55 converts to C-52, only a minor amount of C-55 may serve as a prodrug for C-52, whereas the majority is handled by the biosystem through a different route of elimination. Tissue distribution combined with rate of conversion may be an important determinant of the relative effectiveness of other epoxide-chlorohydrin pairs of cryptophycins.

Cathepsin B is a lysosomal cysteine protease in normal cells and tissues. In malignant tumors and premalignant lesions, the expression of cathepsin B is highly upregulated and the enzyme is secreted and becomes associated with the cell surface. Increases in expression are mediated at many levels ranging from gene amplification to increased stability of mRNA and protein. Cathepsin B is synthesized as a preproenzyme and the primary pathways for its normal trafficking to the lysosome utilize mannose 6-phosphate receptors (MPRs). Inactive procathepsin B is processed to active single and double chain forms of cathepsin B in the late endosomes and lysosomes, respectively. Tumor cells secrete procathepsin B and both active forms of cathepsin B. Secretion of procathepsin B occurs principally as a result of increased expression, whereas secretion of active cathepsin B seems to involve active processes that can be induced by a variety of mechanisms. Once secreted procathepsin B binds to the tumor cell surface via p11, the light chain of the annexin II heterotetramer. This binding seems to facilitate conversion of procathepsin B to its active forms. Cathepsin B and the annexin II heterotetramer colocalize in caveolae (lipid raft) fractions isolated from tumor cells. Serine proteases and matrix metalloproteinases also have been found to associate with caveolae and some with the annexin II heterotetramer. Our working hypothesis is that pericellular cathepsin B through its proximity to other proteases in caveolae participates in, perhaps even initiates, a proteolytic cascade on the tumor cell surface.

The purpose of this study was to determine the outcome of patients receiving external beam radiation for an elevated postprostatectomy prostate-specific antigen (PSA) level. Between December 1991 and September 1998, 108 patients received definitive radiation therapy for elevated postprostatectomy PSA levels. The median dose of irradiation was 68 Gy (range, 48-74 Gy). During treatment, the PSA levels were checked an average of 5 times (range, 3-7 times). Prostate-specific antigen values were judged to decline or increase during treatment if they changed by more than 0.2 ng/mL. After treatment, biochemical failure was defined as a measurable or rising PSA > 0.2 ng/mL. Median follow-up was 51 months (range, 3-112 months). Fifty-eight patients (54%) had evidence of biochemical failure. The 3- and 5-year actuarial biochemical relapse-free (bNED) survivals for all patients were 55% and 39%, respectively. Upon univariate analysis, intratreatment PSA and preradiation PSA were significant predictors of bNED survival. Patients with a PSA level that decreased during treatment had a 5-year bNED survival of 43% compared to 10% in patients with an increasing PSA level (P = 0.0002). Using the preradiation therapy PSA value as a continuous variable, higher preradiation therapy PSA levels were associated with an increased risk of failure (P = 0.004). Cut points of pretreatment PSA were derived at 0.9 ng/mL and 4.2 ng/mL using the Michael Leblanc recursive partitioning algorithm. The 5-year bNED rate for patients with a preradiation therapy PSA < 0.9 ng/mL was 45% versus 42% for patients with preradiation therapy PSA between 0.9 and 4.2 ng/mL and 21% for patients > or = 4.2 ng/mL (P = 0.0003). Patients with a Gleason score of < or = 7 had a 5-year bNED rate of 38% compared to 37% for patients with a Gleason score > 7 (P = 0.27). Other factors examined individually that did not reach statistical significance included time from surgery to radiation therapy, race, seminal vesicle involvement, pathological stage, surgical margin, and perineural invasion. Upon multivariate analysis, only preradiation therapy PSA (P < 0.001) and the PSA trend during radiation therapy (P < 0.001) were significant factors. The results of therapeutic radiation for patients with elevated postprostatectomy PSA levels are sufficiently poor; other strategies should be explored as alternatives, including early adjuvant postprostatectomy irradiation or the use of combined hormonal and radiation therapy in the salvage situation.

Chemoprevention means prevention of cancer by administering chemical compounds. Ideal chemopreventive agents are non-toxic, inexpensive and can be taken orally. A large number of natural and synthetic compounds have cancer preventive properties in cell culture or animal model studies. With the demonstration of tamoxifen's ability to prevent breast cancer in women, the feasibility of chemoprevention in humans has now been firmly established. Current challenge is to build on this success and identify non-toxic chemopreventive agents. A promising area of research is clinical studies with botanicals, cancer preventive compounds in fruits, vegetables and other plants.

Purpose: The prognostic significance of Her-2/neu overexpression in muscle-invasive urothelial carcinoma of the bladder is largely unknown. Accurate determination of Her-2/neu overexpression may have therapeutic importance., Experimental Design: Eighty consecutive cases of muscle-invasive urothelial carcinoma of the bladder treated by radical cystectomy with available follow-up were analyzed. In each case, one representative section was stained with anti-Her-2/neu. Staining was graded as 1 = faint/equivocal, 2 = moderate, and 3 = strong and was considered positive if > or =2. In those cases with a metastasis, the stain was also performed in the metastatic tumor. Results were correlated with survival., Results: Twenty-two (28%) cases were considered Her-2/neu-positive in the primary tumor, and 17 of 32 (53%) were considered Her-2/neu-positive in the lymph node metastasis. Median survival for Her-2/neu-positive primary tumors was 33 months, compared with 50 months for Her-2/neu-negative cases (P = 0.46). Similarly, Her-2/neu overexpression in the lymph node metastasis did not predict survival. Sixty metastatic urothelial carcinomas were further studied by comparing Her-2/neu expression in the primary tumor with that of the lymph node and/or distant metastasis. Forty-five percent of Her-2/neu-negative primary tumors had a Her-2/neu-positive lymph node metastasis, whereas only one case (8%) of Her-2/neu-positive primary tumors was Her-2/neu-negative in the lymph node metastasis (P = 0.009). Similarly, 67% of Her-2/neu-negative primary tumors had a Her-2/neu-positive distant metastasis, whereas no Her-2/neu-positive primary tumor was negative in the metastasis (P = 0.429)., Conclusions: Her-2/neu overexpression in primary or metastatic tumor did not predict survival in this cohort of muscle-invasive tumors. Overexpression in the primary tumors consistently predicts overexpression in a distant or regional metastasis. However, some Her-2/neu-negative primary tumors may show overexpression in their corresponding metastasis. Her-2/neu analysis in a metastasis may be necessary to accurately determine Her-2/neu status in metastatic bladder urothelial carcinoma.

Following high-dose chemotherapy (HDC) and peripheral blood progenitor cell transplantation (PBPCT), there are profound changes in leukocyte homeostasis and the immune system is compromised. Transplantation of purified CD34+ cells may further compromise immune recovery because the grafts are depleted of mature immune cells. However, a detailed monitoring of immune cell reconstitution has not been done. We monitored blood levels of antigen-presenting cells (APC) and of lymphocytes by multi-color flow cytometry at different times post CD34+ PBPCT. We found a rapid normalization of circulating levels of the antigen-presenting CD11c+ dendritic cells (defined as lineage- HLA-DR+ CD11c+ cells). There was a slight over-representation of lin- DR+ CD11c- cells at day 42 post transplantation suggesting that the composition of the APC population might be affected. Normal levels of total T, B and NK lymphocytes were rapidly achieved but the composition of the T cell population was abnormal. Patients had elevated levels of CD8+ T cells at early times and a persistent reduction in levels of naive CD8+ T cells (CD8+ CD4- CD45RA+ CD27+) and of naive CD4+ T cells (CD4+CD3+ CD8- CD45RA+). Thus, we found a rapid recovery of DC after CD34+ PBPCT but the specific numerical defects in naive T cells are likely to be a major cause of immune dysfunction in the patients. Bone Marrow Transplantation (2000) 25, 1249-1255.

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7, a mutant dominant-negative Ikaros protein. In contrast, Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore, Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus, distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137)

Dietary fat and energy intake have been implicated in breast cancer etiology. To examine the relative importance of these dietary factors on markers of cancer risk in women, we designed an intervention trial to selectively decrease fat and/or energy intake in free-living, premenopausal women who were somewhat overweight. The study used a 2 x 2 factorial design to evaluate the independent and interactive effects of dietary fat and energy. The diets were nonintervention, low fat (15% of energy from fat, maintenance of energy intake), low energy (25% energy reduction), and combination low fat and low energy. We utilized an individualized counseling approach with self-selection of foods. Women on the low-fat and combination diets were asked to meet given daily goals for fat grams and food group exchanges, while women on the low-energy diet used only food group exchanges. Of the 113 premenopausal women randomized who were eligible for analysis, 43% were African-American. A total of 88 women completed the 12-week program, and adherence to the dietary goals was similar in both racial groups. Women on the low-fat diet were able to reduce dietary fat intake to 19% of energy by 4 weeks and to 17% by 12 weeks with a slight decrease in energy intake. Women on the low-energy diet met their energy reduction goals by four weeks while maintaining percentage of energy from fat. Women on the combination diet largely met their goals by four weeks as well. These data indicate that it is possible to selectively manipulate dietary fat and energy intake in women over a short period of time, which makes clinical studies on the relative effects of these two dietary variables on cancer risk biomarkers readily feasible.

Although impressive biologic advances have increased understanding of leukemogenesis, we know little about the causes of the acute leukemias. Epidemiologic studies have focused primarily on children. Higher birth weight is associated with an increased risk of childhood acute leukemia. Several theories have been advanced that may account for these observations, and additional biologic studies are needed. Some epidemiologic studies suggest that the acute leukemias in children may have an infectious component. Again, further work, especially in the area of specific causative agents, is necessary. Another area for future epidemiologic study includes investigation of exposure to natural and synthetic DNA topoisomerase II inhibitors. Preliminary evidence suggests that exposure to these agents, which are found in certain foods and medications, may be related to the subsequent development of acute leukemia in infants.

The ability of ionic mercury to induce protein tyrosine phosphorylation in mouse spleen cells and in the mouse WEHI-231 B-cell lymphoma was investigated. We have confirmed previous studies which showed that exposure to high levels (several hundred microM) of mercury lead to very large increases in the level of protein tyrosine phosphorylation in these cell systems. However we have also demonstrated that low levels (in the order of 0.1 to 1.0 microM) of mercury also significantly upregulate protein tyrosine phosphorylation. Mercury induced protein tyrosine phosphorylation is inhibited by the mercury chelator penicillamine and by pretreating treating target cells with the sulfhydryl blocking reagent N-hydroxymaleimide. These results suggest that exposure to low levels of mercury could potentially interfere with lymphocyte signal transduction and so offer a possible explanation as to how mercury exposure could lead to immune cellular dysfunction. On a molecular level, the results suggest that the site(s) of action with respect to mercury dependent induction of protein tyrosine phosphorylation is likely a free disulfide group or groups located on the outer leaflet of the plasma membrane.

Interleukin-2 (IL-2) is a lymphokine produced by T-cells that has a number of immunomodulatory effects. Treatment of metastatic melanoma with recombinant interleukin-2 (rIL-2)-based therapies represents one of the earliest attempts at systemic immunomodulation as a therapy for cancer. Initial studies showed objective response rates with rIL-2 therapy alone in the range of 15 - 20% with some durable responses. A multitude of studies have been undertaken with various rIL-2 regimens, with and without co-administration of lymphokine-activated cells or tumour-infiltrating lymphocytes. However, the optimum dose and treatment schedule for rIL-2-based therapy in metastatic melanoma, remains controversial. There are also no clear immunological parameters that can reliably predict antitumour response to rIL-2-based therapy. Ongoing research remains active in exploring the role of rIL-2 in the therapy of malignant melanoma (MM), particularly in conjunction with cytotoxic therapy.

Flow cytometric analysis of nucleoids is useful to investigate nuclear matrix-DNA associations in cells. We have now applied this technique to examine whether differences in nucleoid structure can be detected between tumor and normal-like human breast epithelial cells. We have previously shown that MCF-7 tumor and MCF-10A normal-like human breast cells exhibit different levels of endogenous oxidative DNA damage as well as differences in response to hydrogen peroxide. We therefore examined whether flow cytometric analysis of nucleoids can be used to detect both endogenous DNA damage and DNA damage induced by hydrogen peroxide in these cells. Nucleoids were prepared by lysis of MCF-7 and MCF-10A cells with a high-salt buffer. The size of the DNA loops around the nuclear matrix core was detected by measuring the forward light-scatter signal in the presence of ethidium bromide. The relaxation and supercoiling of DNA in the presence of low and high concentrations of ethidium bromide, respectively, was similar in MCF-7 tumor and MCF-10A normal-like cells. After treatment of cells with 100 microM and higher of hydrogen peroxide, there was a statistically significant increase in the forward light scatter from nucleoids prepared in the presence of high concentrations of ethidium bromide, and this increase was similar in both cell lines. The changes in the forward light scatter signal with increasing hydrogen peroxide concentration did not mimic the patterns of increases in single strand breaks or formation of 5-hydroxymethyl-2 '-deoxyuridine that we reported previously. This indicates that the flow cytometric technique probably detects other types of changes induced by hydrogen peroxide.

The growth inhibitory properties of alpha-tocopheryl hemisuccinate (vitamin E succinate) and related compounds were examined in MCF-7 human breast tumor cells and MCF-10A normal-like human breast cells since they have been suggested to be an effective antitumor compound. The data showed that both alpha-tocopherol hemisuccinate and a structurally-similar compound, cholesteryl hemisuccinate, inhibited the growth of MCF-7 and MCF-10A cells, while alpha-tocopherol, cholesterol, cholesteryl sulfate and Tris succinate had little effect on cell growth. The ether analogs of the succinate esters, alpha-tocopheryloxybutyric acid and cholesteryloxybutyric acid, also inhibited growth of MCF-7 and MCF-10A cells, indicating that hydrolysis of the succinate esters by esterases is not required for the antiproliferative effects. The antiproliferative effects of these succinate esters and ethers may be related to their physiochemical properties that allow incorporation into cell membranes.

IL-12 is a novel heterodimeric cytokine important for the regulation and differentiation of lymphocytes and NK cells. Like other cytokines, IL-12 mediates its biologic activity through high-affinity receptors expressed on responsive cells. To date, a large number of receptors for IL-12 have been found only on PBMC following activation with PHA or IL-2. To gain further knowledge of the IL-12R complex and the IL-12 signal transduction pathway in cytotoxic T cells, we studied a number of human T cell lines that had been transformed to permanent growth with Herpesvirus saimiri, an oncogenic virus of nonhuman primates. This paper reports the expression of IL-12R on a human gamma delta T cell line that responds to IL-12 with enhanced cytolytic activity and increased expression of cytolytic effector molecules granzyme B and perforin. Using these T cells as a model of IL-12 signal transduction, we confirmed that these events involve members of the Janus kinase family of nonreceptor tyrosine kinases JAK2, TYK2, and signal transducer and activator of transcription 4.