Development of a Novel Imaging Agent for Determining Albumin Uptake in Solid Tumors.

PURPOSE: The purpose of this study was to investigate the albumin-binding compound 111In-C4-DTPA as an imaging agent for the detection of endogenous albumin accumulation in tumors. METHODS: 111In-C4-DTPA was injected in healthy nude mice for pharmacokinetic and biodistribution studies (10 min, 1, 6, 24, and 48 h, n = 4) and subsequently in tumor-bearing mice for single-photon emission computed tomography/X-ray-computed tomography (SPECT/CT) imaging studies. Four different human tumor xenograft models (LXFL529, OVXF899, MAXFTN401, and CXF2081) were implanted subcutaneously unilaterally or bilaterally (n = 4-8). After intravenous administration of 111In-C4-DTPA, SPECT/CT images were collected over 72 h at 4-6 time points. Additionally, gamma counting was performed for the blood, plasma, lungs, heart, liver, spleen, kidneys, muscle, and tumors at 72 h post-injection. RESULTS: 111In-C4-DTPA bound rapidly to circulating albumin upon injection, and the radiolabeled albumin conjugate thus formed was stable in murine and human serum. SPECT/CT images demonstrated a time-dependent uptake with a maximum of 2.7-3.8% ID/cm3 in the tumors at approximately 24 h post-injection and mean tumor/muscle ratios in the range of 3.2-6.2 between 24 and 72 h post-injection. The kidneys and bladder were the predominant elimination organs. Gamma counting at 72 h post-injection showed 1.3-2.5% ID/g in the tumors and mean tumor/muscle ratios in the range of 4.9-9.4. CONCLUSION: 111In-C4-DTPA bound rapidly to circulating albumin upon injection and showed time-dependent uptake in the tumors demonstrating a potential for clinical application as a companion imaging diagnostic for albumin-binding anticancer drugs.

Understanding how non-coding genomic polymorphisms affect gene expression.

The NIH Common Fund GTEx project is designed to serve as a data and post-mortem tissue resource to the research community. The project is testing the role of genomic variation in altering gene expression across a wide array of tissues in a large number of human post-mortem donors. Both data and tissue samples are available to the research community for additional studies.

Course and outcome of fetuses suspected of having coarctation of the aorta during gestation.

PURPOSE: To report the course and outcome of a group of fetuses with prenatal suspicion of coarctation of the aorta. MATERIALS AND METHODS: Retrospective observational study in two tertiary fetal cardiology centers between 1993 - 2005. RESULTS: 96 fetuses of whom 52 infants were born alive were studied. Of the 52 liveborn infants, 34 had coarctation of the aorta (65.4 %), thirteen had prenatally diagnosed additional cardiac anomalies (VSD, ASD, aortic and pulmonary stenosis, persistent left superior vena cava) and three were managed as having hypoplastic left heart syndrome. Three neonates had additional extracardiac malformations diagnosed prenatally. 22 neonates underwent surgery, nineteen within the first ten days of life. One neonate only developed clinical signs of coarctation on the fourteenth day of life. The early surgical mortality was three of 22 (13.6 %). The mortality was influenced by prematurity. The survival rate on the basis of intention-to-treat was twenty-nine of 34 neonates with confirmed coarctation (85.3 %). CONCLUSION: Coarctation of aorta during fetal life continues to be a difficult diagnosis. The potential of progressive hypoplasia of left heart structures during gestation in the case of fetal aortic isthmus stenosis with the development of a hypoplastic left heart should be kept in mind and therefore sequential echo-cardiography is recommended during gestation.

Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation.

Jurkat T cells showed a major, early decrease in blue autofluorescence in response to Fas/Apo-1/CD95 cross-linking or stimulation with cell-permeant ceramide. This indicates the oxidation/depletion of NADH or NADPH before the onset of apoptosis. Kinetic studies, cytofluorimetric multiparameter analyses and cell sorting experiments indicated a close temporal relationship between NAD(P)H oxidation/depletion and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). In contrast, NAD(P)H depletion was detected well before several other changes associated with late apoptosis, including enhanced superoxide generation, phosphatidylserine exposure on the cell surface, loss of cytosolic K(+), decreased cytoplasmic pH, nuclear DNA fragmentation, cell shrinkage, loss of viability and the appearance of the mitochondrial antigen APO2.7. Full activation of caspase 9 and caspase 3 appeared to be correlated with the appearance of superoxide anions in the mitochondria, and followed the drop in NADPH. Overexpression of the apoptosis-inhibitory proto-oncogene Bcl-2, which encodes an inhibitor of the mitochondrial permeability transition (PT) pore, delayed both the DeltaPsi(m) disruption and the depletion of NAD(P)H. Similar effects were observed with the pharmacological PT pore inhibitors, bongkrekic acid and cyclosporin A. Thus there appears to be a close functional relationship between mitochondrial and cellular redox changes during early apoptosis; events that are inhibited by Bcl-2.

Intracellular markers.

An increased level of complexity will be encountered when developing protocols for intracellular markers. Protocols for surface markers have been successfully standardized, however it is understood that no single method is appropriate for all intracellular staining. A systematic approach should be followed, including knowledge of antigen location and functional state, selection of cell fixative and cell permeabilizer, antibody specificity and class/subclass, fluorochrome, fluorochrome to protein ratio (F:P), and use of adequate controls, including isotype-matched negative controls and positive and negative cell controls. Even though it is impossible to recommend a single technique to stain all intracellular antigens, the authors present a logical approach to follow when developing a staining protocol.

Active heroin injectors' perceptions and use of methadone maintenance treatment: cynical performance or self-prescribed risk reduction?

In addition to the numerous heroin users who voluntarily enter methadone treatment as a way to free themselves from illicit drug addiction and those ordered to do so by the courts, there are a large number of opioid users who enter methadone treatment with other objectives in mind. These include shorter-term goals that users do not necessarily equate with complete heroin abstinence. In this paper we report the results of a qualitative study designed to identify and describe the motivations active heroin users have for entering methadone treatment, and to suggest that many of these short-term methadone episodes may operate as self-prescribed attempts at risk reduction, and act as pilot tests for users considering or anticipating entering treatment to quit the use of illicit drugs. We argue that heroin users' motivations, perceptions about methadone, and the strategies they devise for adapting methadone treatment for their own needs should be recognized for their value in reducing the multiple risks associated with drug use.

Differentiation and assessment of cell death.

Three documented cell death pathways, apoptosis, necrosis, and oncosis will be discussed. The end result of each pathway is cell death; however, the path by which death is achieved and the morphological and physiological traits of each may be strikingly distinct. Now that well characterized models have been established for particularly apoptosis, the induction pathway(s) has received much attention and the pathway pathology is beginning to be understood. Three model systems were investigated: APO-1/Fas, hypoxia, and oncosis. Cell death was induced, and during a time course sampling, a variety of methodologies, including DNA fragmentation by flow cytometry and gel electrophoresis, DNA staining, flow cytometric light scatter, transmission electron microscopy, anti-tubulin, Trypan blue, annexin V, and anti-APO2.7 were employed to monitor the cell death progress. The apoptotic pathway in the CD95-induced Jurkat cell model was further investigated using caspase inhibitor peptides and analyzed for APO2.7 antigen expression and DNA fragmentation by flow cytometry. Time course sampling characterized the cell death pathway and helped to differentiate the capabilities of the methods. The time to response and duration of the response were dependent upon cell type and method of induction. The CD95-induced Jurkat cell model showed a classical apoptotic response; however the MDA-MB-175-VII hypoxia model and the anti-5A9 induced oncosis model were not as clear. Each methodology shows advantages and disadvantages that allow the investigator to select several methods to identify, monitor, and enumerate cells with respect to cell death progression using time course studies.

APO2.7 defines a shared apoptotic-necrotic pathway in a breast tumor hypoxia model.

A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.

Mitoguazone induces apoptosis via a p53-independent mechanism.

Mitoguazone (methylglyoxal bisguanylhydrazone, methyl-GAG or MGBG) is a synthetic polycarbonyl derivative with activity in patients with Hodgkin's and non-Hodgkin's lymphoma, head and neck cancer, prostate cancer, and esophageal cancer. Mitoguazone has also recently been documented to have activity in patients with AIDS-related lymphoma. Among anticancer drugs, mitoguazone has a unique mechanism of action via interference with the polyamine biosynthetic pathway. Polyamines stabilize DNA structure by non-covalent cross-bridging between phosphate groups on opposite strands. In addition, mitoguazone causes uncoupling of oxidative phosphorylation. In this study, the ability of mitoguazone to induce apoptosis by inhibiting the polyamine pathway was assessed in three Burkitt's lymphoma cell lines (Raji, Ramos and Daudi) and one prostate carcinoma cell line (MPC 3). Additional evaluations were performed in two human breast cancer cell lines (MCF7 with wild-type p53 and VM4K with mutated p53) to determine whether the p53 tumor suppressor gene was required for efficient apoptosis induction. The present study demonstrated that mitoguazone induces apoptosis in all the different human cancer cell lines tested in a concentration- and time-dependent way, and triggers a p53-independent programmed cell death in the human breast cancer MCF7 cell line.

HIV risk behaviors associated with the injection process: multiperson use of drug injection equipment and paraphernalia in injection drug user networks.

This study examines drug acquisition and multiperson use of paraphernalia, drugs, and needles/syringes. Ethnographers observed 54 injection episodes in which IDUs were linked by HIV risk behaviors, and developed a typology of higher-risk, lower-risk, and nonsharing-risk networks. Multiperson use of injection paraphernalia or drug solution occurred in most injection events (94%). Serial use of syringes/needles occurred infrequently (14%) relative to "backloading" (37%) and reuse of paraphernalia (cookers 84%, cotton 77%, water 77%). Higher-risk injection networks were characterized by larger size and pooling of resources for drugs. Prevention messages must include avoiding reuse of injection paraphernalia and transfer of drug solution.

A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors.

A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.

Directed growth of early cortical axons is influenced by a chemoattractant released from an intermediate target.

Projection neurons throughout the mature mammalian neocortex extend efferent axons either through the ventrolaterally positioned internal capsule to subcortical targets or through the dorsally located midline corpus callosum to the contralateral cortex. In rats, the internal capsule is pioneered on E14, but the corpus callosum is not pioneered until E17, even though these two types of projection neurons are generated at the same time. Here we use axonal markers to demonstrate that early cortical axon growth is directed toward the nascent internal capsule, which could account for the timing difference in the development of the two efferent pathways. This directed axon growth may be due to a chemoattractant and/or a chemorepellent secreted by intermediate targets of cortical efferent axons, the nascent internal capsule, or the medial wall of the dorsal telencephalon (MDT), respectively. To test for these soluble activities, explants of E15 rat neocortex and intermediate targets were cocultured in collagen gels. Cortical axon outgrowth was directed toward the internal capsule, but outgrowth was nondirected and suppressed when cocultured with MDT, suggesting that the internal capsule releases a chemoattractant for cortical axons, whereas the MDT releases a chemosuppressant. Because the chemoattractant Netrin-1 is expressed in the internal capsule, we cocultured cortical explants with E13 rat floor plate, which expresses Netrin-1, or with Netrin-1-transfected or control-transfected 293T cells. Cortical axon growth was directed toward both floor plate and Netrin-1-transfected 293T cells, as it had been toward the internal capsule, but not toward control-transfected 293T cells. These findings suggest that early events in cortical axon pathfinding may be controlled by a soluble activity which attracts initial axon growth toward the internal capsule and that this activity may be due to Netrin-1.

Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue.

To understand how the skeletal muscle lineage is induced during vertebrate embryogenesis, we have sought to identify the regulatory molecules that mediate induction of the myogenic regulatory factors MyoD and Myf-5. In this work, we demonstrate that either signals from the overlying ectoderm or Wnt and Sonic hedgehog signals can induce somitic expression of the paired box transcription factors, Pax-3 and Pax-7, concomitant with expression of Myf-5 and prior to that of MyoD. Moreover, infection of embryonic tissues in vitro with a retrovirus encoding Pax-3 is sufficient to induce expression of MyoD, Myf-5, and myogenin in both paraxial and lateral plate mesoderm in the absence of inducing tissues as well as in the neural tube. Together, these findings imply that Pax-3 may mediate activation of MyoD and Myf-5 in response to muscle-inducing signals from either the axial tissues or overlying ectoderm and identify Pax-3 as a key regulator of somitic myogenesis.

Differential regulation of cell cycle characteristics and apoptosis in MMTV-myc and MMTV-ras mouse mammary tumors.

We have used the MMTV-myc and MMTV-ras transgenic mouse mammary tumor models (T. A. Stewart et al., Cell, 38: 627-637, 1984, and E. Sinn et al., Cell, 49: 465-475, 1987) to evaluate how the c-myc and v-Ha-ras oncogenes influence tumor growth characteristics in vivo. MMTV-myc tumors had much higher levels of spontaneous apoptosis than MMTV-ras tumors, whereas intermediate levels were observed in MMTV-myc/ras tumors. Significant differences in cell cycle characteristics were also observed in tumors from mice of the three genotypes. Tumors from MMTV-myc mice had lower G1 and higher S-phase fractions than MMTV-ras tumors, with intermediate values again observed in the MMTV-myc/ras tumors. Despite these differences, however, tumor growth rates for the different groups were similar. These findings highlight the importance of the balance between cell cycle regulation and cell death in determining the kinetics of tumor growth and indicate that distinct oncogenes can have a profound influence on that balance.

Increased tumor proliferation and genomic instability without decreased apoptosis in MMTV-ras mice deficient in p53.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53(-/-) mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53(+/+), ras/p53(+/-), or ras/p53(-/-)). ras/p53(-/-) mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53(+/+) and ras/p53(+/-) mice were of mammary origin, ras/p53(-/-) mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53(-/-) mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53(+/+) mice. Interestingly, the increased growth rates of ras/p53(-/-) tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.