Smoothed Nested Testing on Directed Acyclic Graphs.

We consider the problem of multiple hypothesis testing when there is a logical nested structure to the hypotheses. When one hypothesis is nested inside another, the outer hypothesis must be false if the inner hypothesis is false. We model the nested structure as a directed acyclic graph, including chain and tree graphs as special cases. Each node in the graph is a hypothesis and rejecting a node requires also rejecting all of its ancestors. We propose a general framework for adjusting node-level test statistics using the known logical constraints. Within this framework, we study a smoothing procedure that combines each node with all of its descendants to form a more powerful statistic. We prove a broad class of smoothing strategies can be used with existing selection procedures to control the familywise error rate, false discovery exceedance rate, or false discovery rate, so long as the original test statistics are independent under the null. When the null statistics are not independent but are derived from positively-correlated normal observations, we prove control for all three error rates when the smoothing method is arithmetic averaging of the observations. Simulations and an application to a real biology dataset demonstrate that smoothing leads to substantial power gains.

Interaction of MYC with host cell factor-1 is mediated by the evolutionarily conserved Myc box IV motif.

The MYC family of oncogenes encodes a set of three related transcription factors that are overexpressed in many human tumors and contribute to the cancer-related deaths of more than 70,000 Americans every year. MYC proteins drive tumorigenesis by interacting with co-factors that enable them to regulate the expression of thousands of genes linked to cell growth, proliferation, metabolism and genome stability. One effective way to identify critical co-factors required for MYC function has been to focus on sequence motifs within MYC that are conserved throughout evolution, on the assumption that their conservation is driven by protein-protein interactions that are vital for MYC activity. In addition to their DNA-binding domains, MYC proteins carry five regions of high sequence conservation known as Myc boxes (Mb). To date, four of the Mb motifs (MbI, MbII, MbIIIa and MbIIIb) have had a molecular function assigned to them, but the precise role of the remaining Mb, MbIV, and the reason for its preservation in vertebrate Myc proteins, is unknown. Here, we show that MbIV is required for the association of MYC with the abundant transcriptional coregulator host cell factor-1 (HCF-1). We show that the invariant core of MbIV resembles the tetrapeptide HCF-binding motif (HBM) found in many HCF-interaction partners, and demonstrate that MYC interacts with HCF-1 in a manner indistinguishable from the prototypical HBM-containing protein VP16. Finally, we show that rationalized point mutations in MYC that disrupt interaction with HCF-1 attenuate the ability of MYC to drive tumorigenesis in mice. Together, these data expose a molecular function for MbIV and indicate that HCF-1 is an important co-factor for MYC.

A common functional consequence of tumor-derived mutations within c-MYC.

The relevance of changes to the coding sequence of the c-MYC oncogene to malignancy is controversial. Overexpression of a pristine form of MYC is observed in many cancers and is sufficient to drive tumorigenesis in most contexts. Yet missense changes to MYC are found in ~50% of Burkitt's lymphomas, aggregate within an amino-terminal degron important for proteasomal destruction of MYC, and where examined profoundly enhance the tumorigenic properties of MYC in vitro and in vivo. Much of the controversy surrounding these mutants stems from the limited number of mutations that have been evaluated and their clustering within a single region of the MYC protein; the highly-conserved Myc box I (MbI) element. Here, by analysis of extant genomic data sets, we identify a previously unrecognized hotspot for tumor-associated MYC mutations, located in a conserved central portion of the protein. We show that, despite their distal location in MYC, mutations in this region precisely phenocopy those in MbI in terms of stability, in vitro transformation, growth-promoting properties, in vivo tumorigenesis and ability to escape p53-dependent tumor surveillance mechanisms. The striking parallels between the behavior of tumor-derived mutations in disparate regions of the MYC protein reveals that a common molecular process is disrupted by these mutations, implying an active role for these mutations in tumorigenesis and suggesting that different therapeutic strategies may be needed for treatment of lymphomas expressing wild type versus mutant forms of MYC protein.

Transcriptional control and the ubiquitin-proteasome system.

Regulation of transcription is a critically important process that controls development, differentiation, and the maintenance of cellular homeostasis. Cells have evolved numerous mechanisms to keep gene transcription tightly in check, some of which involve the ubiquitin-proteasome system. In this chapter, we review evidence supporting the concept that ubiquitin and the proteasome not only control transcription, but provide the biochemical means to drive key steps in the transcription process forward.

Synthesis and characterization of branched poly(L-glutamic acid) as a biodegradable drug carrier.

Polymeric drug delivery systems are used not only to improve aqueous solubility of drug molecules but also to achieve desirable pharmacokinetics and an enhanced therapeutic index. New biodegradable polymers are needed to improve the biodistribution and targeting-ability of polymeric carriers. In this study, the synthesis and characterization of branched poly(L-glutamic acid) (PG) containing multiple PG chains centered on a poly(amidoamine) (PAMAM) dendrimer or polyethyleneimine (PEI) cores were described. The branched PG polymers were obtained by ring-opening polymerization of benzyl ester of L-glutamic acid N-carboxyanhydride using PAMAM or PEI as the initiator. These polymers were degradable in the presence of the lysosomal enzyme cathepsin B, albeit more slowly than linear PG. Unlike conventional linear PG, each branched PG possessed multiple terminal amino groups. This made it possible to attach multiple targeting moieties selectively to the termini of branched PG. Conjugation of monofunctional or heterodifunctional polyethylene glycol to the chain ends of branched PG was demonstrated in the presence of side chain carboxyl groups. Furthermore, folic acid, a model targeting moiety, and the near-infrared dye indocyanine green, a model diagnostic agent, were successfully conjugated to the terminal amino groups and the side chain carboxyl groups of branched PG, respectively. The resulting conjugate had reduced nonspecific interaction and bound selectively to tumor cells expressing folate receptors. Thus, branched PG may be useful as a polymeric carrier for targeted drug delivery.

Regulation of transcriptional activation domain function by ubiquitin.

The ability of transcriptional activation domains (TADs) to signal ubiquitin-mediated proteolysis suggests an involvement of the ubiquitin-proteasome pathway in transcription. To probe this involvement, we asked how ubiquitylation regulates the activity of a transcription factor containing the VP16 TAD. We show that the VP16 TAD signals ubiquitylation through the Met30 ubiquitin-ligase and that Met30 is also required for the VP16 TAD to activate transcription. The requirement for Met30 in transcription is circumvented by fusion of ubiquitin to the VP16 activator, demonstrating that activator ubiquitylation is essential for transcriptional activation. We propose that ubiquitylation regulates TAD function by serving as a dual signal for activation and activator destruction.

Transcriptional activation: risky business.

Transcriptional regulation is all about getting RNA polymerase to the right place on the gene at the right time and making sure that it is competent to conduct transcription. Traditional views of this process place most of their emphasis on the events that precede initiation of transcription. We imagine a promoter-bound transcriptional activator (or collection of activators) recruiting components of the basal transcriptional machinery to the DNA, eventually leading to the recruitment of RNA polymerase II and the onset of gene transcription. Although these events play a crucial role in regulating gene expression, they are only half the story. Correct regulation of transcription requires that polymerase not only initiates when and where it should, but that it stops initiating when no longer appropriate. But how are the signals from transcriptional activators, telling RNA polymerase to fire, terminated? Is this process governed by chance, with activators simply falling off the promoter at a certain frequency? Or is there some more direct mechanism, whereby activators are aggressively limited from uncontrolled promoter activation? A new article by suggests the latter may be true, and provides a mechanism for how a component of the basal transcription machinery can mark the activators it has encountered, sentencing them to an early death or banishing them from the nucleus. The ability of the basal transcriptional apparatus to mark activators provides an efficient way to limit activator function and ensures that continuing transcription initiation at a promoter is coupled to the continuing synthesis and activation of transcriptional activators.

In vivo and in vitro measurement of apoptosis in breast cancer cells using 99mTc-EC-annexin V.

OBJECTIVE: The purpose of this study was to develop an imaging technique to measure and monitor tumor cells undergoing programmed death caused by radiation and chemotherapy using 99mTc-EC-annexin V. Annexin V has been used to measure programmed cell death both in vitro and in vivo. Assessment of apoptosis would be useful to evaluate the efficacy and mechanisms of therapy and disease progression or regression. METHODS: Ethylenedicysteine (EC) was conjugated to annexin V using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl as coupling agents. The yield of EC-annexin V was 100%. In vitro cellular uptake, pre- and post-radiation (10-30 Gy) and paclitaxel treatment, was quantified using 99mTc-EC-annexin V. Tissue distribution and planar imaging of 99mTc-EC-annexin V were determined in breast tumor-bearing rats at 0.5, 2, and 4 hrs. To demonstrate in vivo cell apoptosis that occurred during chemotherapy, a group of rats was treated with paclitaxel and planar imaging studies were conducted at 0.5-4 hrs. Computer outlined region of interest (ROI) was used to quantify tumor uptake on day 3 and day 5 post-treatment. RESULTS: In vitro cellular uptake showed that there was significantly increased uptake of 99mTc-EC-annexin V after irradiation (10-30 Gy) and paclitaxel treatment. In vivo biodistribution of 99mTc-EC-annexin in breast tumor-bearing rats showed increased tumor-to-blood, tumor-to-lung and tumor-to-muscle count density ratios as a function of time. Conversely, tumor-to-blood count density ratios showed a time-dependent decrease with 99mTc-EC in the same time period. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-annexin. There was a significant difference of ROI ratios between pre- and post-paclitaxel treatment groups at 2 and 4 hrs post injection. CONCLUSION: The results indicate that apoptosis can be quantified using 99mTc-EC-annexin and that it is feasible to use 99mTc-EC-annexin to image tumor apoptosis.

Effectiveness of water soluble poly(L-glutamic acid)-camptothecin conjugate against resistant human lung cancer xenografted in nude mice.

A camptothecin (CPT) formulation that can be easily administered, is less toxic, and has greater antitumor effect is needed. In this study, a water-soluble CPT derivative was obtained by direct coupling of CPT to poly(L-glutamic acid) (PG) through the C20(S)-hydroxyl group. CPT was released from the resulting conjugate, PG-CPT, in phosphate-buffered saline with a zero-order kinetics in the initial 50 days. The release rates were 0.623% per day, 1.081% per day, and 1.396% per day at pH 5.3, 7.4, and 9.0, respectively. In vitro, PG-CPT was less potent in inhibiting cell growth than was free CPT in all human tumor cell lines tested. However, PG-CPT showed better antitumor activity and tolerability than did CPT in vivo. When H322 human lung tumor cells were inoculated subcutaneously in nude mice, PG-CPT delayed the growth of these well-established tumors with an absolute growth delay of 32 days when given as 4 doses with 4-day intervals between injections at an equivalent CPT dose of 40 mg/kg. When H322 cells were inoculated intratracheally in nude mice, 5 doses of intravenous injection of PG-CPT at an equivalent CPT dose of 10 mg/kg on days 4, 8, 12, 16, and 20 after inoculation significantly prolonged the median survival of treated mice, averaging 1.8-fold that of untreated mice (p=0.01). Increasing the dose of PG-CPT to an equivalent CPT dose of 40 mg/kg per injection administered in 4 doses on days 4, 8, 12, and 16 prolonged the median survival of treated mice by 4-fold (p=0.0008). Significantly, mice with intratracheally inoculated H322 tumors were resistant to both CPT and cisplatin treatments. These studies demonstrated that PG may be used as an effective solubilizing carrier for CPT and that PG-CPT may have potential application in the treatment of lung cancer.

Potentiation of ovarian OCa-1 tumor radioresponse by poly (L-glutamic acid)-paclitaxel conjugate.

PURPOSE: It has been shown that paclitaxel (TXL) can strongly enhance tumor cells' sensitivity to radiation. We examined whether the radiosensitizing effect of paclitaxel can be further enhanced when it is delivered systemically as a polymer-drug conjugate that provides enhanced tumor uptake and prolonged release of TXL in the tumor. METHODS AND MATERIALS: C3Hf/Kam mice bearing 8-mm murine ovarian OCa-1 tumors were treated with i.v.-injected Poly(L-glutamic acid)-paclitaxel (PG-TXL) at an equivalent TXL dose of 80 mg/kg, followed 24 h later by single doses of local radiation ranging from 5 to 15 Gy. To determine how long the radiopotentiation persisted at extended times after PG-TXL administration, mice with OCa-1 tumors were given i.v. PG-TXL and 4, 24, 48, 72, 120, or 168 h later their tumors were irradiated at a dose of 10 Gy. Antitumor activity was determined by delay in tumor growth. Cell cycle distribution was assayed using flow cytometry. Tumor vascular volume was estimated using Tc-99 m-labeled red blood cells. RESULTS: PG-TXL strongly potentiated the radioresponse of the OCa-1 tumor. The enhancement factors ranged from 2.79 to 4.28, depending on radiation dose, when PG-TXL preceded radiation by 24 h. The enhancement factor derived from radiation dose-response curves was as high as 5.13. The radiosensitizing effect of PG-TXL was also dependent on the interval between PG-TXL administration and radiation delivery, with greater enhancement been observed when the interval was decreased. The percentage of G2/M cells was significantly increased to 21.4% 48 h after PG-TXL but declined to a preinjection level of 14.8% 72 h after PG-TXL. PG-TXL only moderately increased the tumor vascular volume by 37% 24 h after PG-TXL administration. CONCLUSION: PG-TXL markedly potentiated response of OCa-1 tumor to radiation. When compared to literature data obtained from the same tumor model used here, PG-TXL exhibited stronger radiosensitization effect than TXL. Although its action is possibly mediated by arrest of cells in G2/M phases of cell cycle and by increased tumor blood supply, PG-TXL may exert its radiopotentiation activity through increased tumor uptake of PG-TXL and sustained release of TXL in the tumor. Our results show that conjugation of TXL to a polymer has the potential to further enhance its radiosensitizing activity and that clinical trials of PG-TXL in combination with radiation is warranted.

Tumor irradiation enhances the tumor-specific distribution of poly(L-glutamic acid)-conjugated paclitaxel and its antitumor efficacy.

The poly(L-glutamic acid)-paclitaxel (PG-TXL) conjugate has been shown to exhibit significantly greater antitumor activity than conventionally formulated paclitaxel (TXL) against solid tumors (Li et al., Cancer Res., 58: 2404-2409, 1998). Here we report that local tumor irradiation enhanced the distribution of PG-TXL given 24 h later to ovarian OCa-1 carcinoma implanted i.m. in C3Hf/Kam mice. Radiation significantly increased tumor uptake of PG-TXL and tumor vascular permeability, caused elevation of the serum concentration of vascular endothelial growth factor, and arrested OCa-1 cells in the G1 phase of cell cycle. The enhancement factors, as measured by incremental tumor growth delay compared with PG-TXL alone, ranged from 1.36-4.44. Complete tumor regression was also observed at a higher radiation dose (>10 Gy) and a higher PG-TXL dose (>80 mg equivalentTXL/kg). Furthermore, combined radiation and PG-TXL produced a significantly greater tumor growth delay than treatment with radiation and TXL when both drugs were given at the same equivalent TXL dose of 60 mg/kg 24 h after tumor irradiation (enhancement factors, 4.44 versus 1.50). These data suggest that conjugation of TXL to poly(L-glutamic acid) is necessary for improved response and that the supra-additive effect of combined radiation and PG-TXL therapy is due in part to modulation of the enhanced permeability and retention effect of macromolecules by radiation. We propose a treatment strategy combining radiation and macromolecular chemotherapy that may have important clinical implications in terms of scheduling and optimization of the therapeutic ratio.

Functional overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis.

Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins destroyed by ubiquitin-mediated proteolysis. In a previous study of sequences targeting the transcription factor Myc for destruction, we observed that the region in Myc signaling ubiquitin-mediated proteolysis overlaps closely with the region in Myc that activates transcription. Here, we present evidence that the overlap of these two activities is not unique to Myc, but reflects a more general phenomenon. We show that a similar overlap of activation domains and destruction elements occurs in other unstable transcription factors and report a close correlation between the ability of an acidic activation domain to activate transcription and to signal proteolysis. We also show that destruction elements from yeast cyclins, when tethered to a DNA-binding domain, activate transcription. The intimate overlap of activation domains and destruction elements reveals an unexpected convergence of two very different processes and suggests that transcription factors may be destroyed because of their ability to activate transcription.

HAM: a new epitope-tag for in vivo protein labeling.

The ability to metabolically label proteins with 35S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of 35S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.

Synthesis of [99mTc]ethylenedicysteine-colchicine for evaluation of antiangiogenic effect.

Angiogenesis is in part responsible for tumor growth and the development of metastasis. Radiolabeled angiongenesis inhibitors would be useful to assess tumor microvasculature density. Colchicine (COL), a potent antiangiogenic agent, is known to inhibit microtubule polymerization and cell arrest at metaphase. This study aimed to develop 99mTc-labeled COL (EC-COL) using ethylenedicysteine (EC) as a chelator to assess tumor microvascular density. EC was conjugated to trimethylcolchicinic acid using N-hydroxysuccinimide and 1-ethyl-3-dimethylaminopropyl carbodiimide as coupling agents with a yield of 50-60%. In vivo stability was analyzed in rabbit serum at 0.5-4 h. Tissue distribution and planar imaging studies of [99mTc]EC-COL were evaluated in breast tumor-bearing rats at 0.5, 2 and 4 h. The data was compared to that using [99mTc]EC (control). The radiochemical yield of [99mTc]EC-COL was greater than 95%. [99mTc]EC-COL was stable in rabbit serum. In vivo biodistribution of [99mTc]EC-COL in breast tumor-bearing rats showed increased tumor-to-blood (0.52+/-0.12 to 0.72+/-0.07) and tumor-to-muscle (3.47+/-0.40 to 7.97+/-0.93) ratios as a function of time. Conversely, tumor-to-blood values showed a time-dependent decrease with [99mTc]EC over the same time period. Planar images confirmed that the tumors could be visualized clearly with [99mTc]EC-COL from 0.5 to 4 h. [99mTc]EC-COL may be useful to assess antiangiogenic and therapeutic effects during chemotherapy.

Enhanced circulatory parameters of human platelets cryopreserved with second-messenger effectors: an in vivo study of 16 volunteer platelet donors.

Platelet transfusion represents an important component of the therapy for thrombocytopenic patients. Prolonged storage capabilities for platelets would alleviate many problems associated with blood banking. Unfortunately, current cryopreservation methods are complex to implement and result in loss of cell number and functional activity. Previous in vitro studies have shown that the use of ThromboSolTM, a platelet-stabilizing formulation, in the cryopreservation of platelets results in significant retention of cell number and in vitro functional activities in addition to reducing the DMSO requirement to only 2%. We evaluated the in vivo circulatory parameters of platelets cryopreserved with ThromboSol. Single donor platelet units were obtained from healthy volunteers (n = 16); the units were then split and cryopreserved with either ThromboSol and 2% DMSO or 6% DMSO alone. Following storage at -80 degrees C for 7-10 d the samples were thawed, washed and radiolabelled with either 51Cr or 111In. The paired samples were then mixed and reinfused into the autologous volunteer. At various time intervals following transfusion a blood sample was drawn and the quantity of circulating labelled platelets was determined. The percent recovery and survival time was determined by multiple-hit analysis. The ThromboSol-treated platelets, as compared to the 6% DMSO-treated platelets, displayed statistically higher percent recovery (40.2% v 28.8%) and survival time (166.3 h v 152.1 h). These results demonstrated that platelets cryopreserved with ThromboSol displayed superior in vitro and in vivo characteristics as compared to the standard 6% DMSO method. The use of ThromboSol allowed for a 3-fold reduction in the DMSO concentration in conjunction with a 40% increase in circulating cell number and normal survival times.

Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc.

The human proto-oncogene c-myc encodes a highly unstable transcription factor that promotes cell proliferation. Although the extreme instability of Myc plays an important role in preventing its accumulation in normal cells, little is known about how Myc is targeted for rapid destruction. Here, we have investigated mechanisms regulating the stability of Myc. We show that Myc is destroyed by ubiquitin-mediated proteolysis, and define two elements in Myc that oppositely regulate its stability: a transcriptional activation domain that promotes Myc destruction, and a region required for association with the POZ domain protein Miz-1 that stabilizes Myc. We also show that Myc is stabilized by cancer-associated and transforming mutations within its transcriptional activation domain. Our data reveal a complex network of interactions regulating Myc destruction, and imply that enhanced protein stability contributes to oncogenic transformation by mutant Myc proteins.

Synthesis, biodistribution and imaging properties of indium-111-DTPA-paclitaxel in mice bearing mammary tumors.

UNLABELLED: Paclitaxel, an antineoplastic agent that stabilizes microtubules and arrests cells in the G2/M cell cycle phase, has shown activity against many common cancers, including ovarian and breast tumors. In order to evaluate the potential value of radiolabeled paclitaxel as an imaging tool in tumors, we synthesized 111In-DEPA-paclitaxel and investigated its biodistribution and gamma scintigraphic imaging properties. METHODS: Mice bearing a paclitaxel-responsive mammary tumor (MCA-4) were used. DTPA-paclitaxel was labeled with 111In with a radiochemical yield of 84% and radiochemical purity of 90%. Each mouse received 5 microCi of radiotracers intravenously for biodistribution studies and 100 microCi for gamma scintigraphic studies. Indium-111-DTPA was used as a control. RESULTS: In tumor-bearing mice, 111In-DTPA was characterized by rapid clearance from the plasma with negligible retention in the tumor, the liver and other body parts. In contrast, 111In-DTPA-paclitaxel exhibited a pharmacological profile resembling that of paclitaxel. Furthermore, a significant uptake of 111In-DTPA-paclitaxel was observed in the tumor. The tumor-to-muscle ratios were 2.64, 3.16 and 6.94 at 30 min, 2 hr and 24 hr, respectively, although absolute uptake in the tumor decreased from 1.95% (injected dose/g) at 30 min to 0.21% at 24 hr after injection. The tumor-to-blood ratio reached 50 at 24 hr after injection. Gamma scintigraphy and autoradiographic studies clearly showed the retention of radiolabeled paclitaxel in the tumor 24 hr after injection. CONCLUSION: These studies suggest that 111In-DTPA-paclitaxel may be clinically useful in studying the uptake of paclitaxel in solid tumors.