Chimeric anti-angiogenin antibody cAb 26–2F inhibits the formation of human breast cancer xenografts in athymic mice

Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26–2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26–2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26–2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26–2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26–2F. Furthermore, the capacities of cAb 26–2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26–2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.

R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

A thyroid hormone receptor coactivator negatively regulated by the retinoblastoma protein

The retinoblastoma protein (Rb) plays a critical role in cell proliferation, differentiation, and development. To decipher the mechanism of Rb function at the molecular level, we have systematically characterized a number of Rb-interacting proteins, among which is the clone C5 described here, which encodes a protein of 1,978 amino acids with an estimated molecular mass of 230 kDa. The corresponding gene was assigned to chromosome 14q31, the same region where genetic alterations have been associated with several abnormalities of thyroid hormone response. The protein uses two distinct regions to bind Rb and thyroid hormone receptor (TR), respectively, and thus was named Trip230. Trip230 binds to Rb independently of thyroid hormone while it forms a complex with TR in a thyroid hormone-dependent manner. Ectopic expression of the protein Trip230 in cells, but not a mutant form that does not bind to TR, enhances specifically TR-dependent transcriptional activity. Coexpression of wild-type Rb, but not mutant Rb that fails to bind to Trip230, inhibits such activity. These results not only identify a coactivator molecule that modulates TR activity, but also uncover a role for Rb in a pathway that responds to thyroid hormone.

Essential role of POU–domain factor Brn-3c in auditory and vestibular hair cell development

The Brn-3 subfamily of POU–domain transcription factor genes consists of three highly homologous members—Brn-3a, Brn-3b, and Brn-3c—that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes plays distinctive roles in the somatosensory, visual, and auditory/vestibular systems.

Tumor necrosis factor-α induces adhesion molecule expression through the sphingosine kinase pathway

The signaling pathways that couple tumor necrosis factor-α (TNFα) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFα induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFα resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFα on endothelial cells leading to extracellular signal-regulated kinases and NF-κB activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFα-induced extracellular signal-regulated kinases and NF-κB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFα-induced endothelial cell activation.

Identification and classification of p53-regulated genes

Sequence-specific transactivation by p53 is essential to its role as a tumor suppressor. A modified tetracycline-inducible system was established to search for transcripts that were activated soon after p53 induction. Among 9,954 unique transcripts identified by serial analysis of gene expression, 34 were increased more than 10-fold; 31 of these had not previously been known to be regulated by p53. The transcription patterns of these genes, as well as previously described p53-regulated genes, were evaluated and classified in a panel of widely studied colorectal cancer cell lines. “Class I” genes were uniformly induced by p53 in all cell lines; “class II” genes were induced in a subset of the lines; and “class III” genes were not induced in any of the lines. These genes were also distinguished by the timing of their induction, their induction by clinically relevant chemotherapeutic agents, the absolute requirement for p53 in this induction, and their inducibility by p73, a p53 homolog. The results revealed substantial heterogeneity in the transcriptional responses to p53, even in cells derived from a single epithelial cell type, and pave the way to a deeper understanding of p53 tumor suppressor action.

The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulated in vivo by an inducible tissue-specific enhancer

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ± 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% ± 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.

Proteasome-dependent degradation of the human estrogen receptor

In eukaryotic cells, the ubiquitin–proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin–proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor β proteins.

Selenium redox biochemistry of zinc–sulfur coordination sites in proteins and enzymes

Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a dozen selenoproteins. We have chosen to examine zinc–sulfur centers as possible targets of selenium redox biochemistry. Selenium compounds release zinc from zinc/thiolate-coordination environments, thereby affecting the cellular thiol redox state and the distribution of zinc and likely of other metal ions. Aromatic selenium compounds are excellent spectroscopic probes of the otherwise relatively unstable functional selenium groups. Zinc-coordinated thiolates, e.g., metallothionein (MT), and uncoordinated thiolates, e.g., glutathione, react with benzeneseleninic acid (oxidation state +2), benzeneselenenyl chloride (oxidation state 0) and selenocystamine (oxidation state −1). Benzeneseleninic acid and benzeneselenenyl chloride react very rapidly with MT and titrate substoichiometrically and with a 1:1 stoichiometry, respectively. Selenium compounds also catalyze the release of zinc from MT in peroxidation and thiol/disulfide-interchange reactions. The selenoenzyme glutathione peroxidase catalytically oxidizes MT and releases zinc in the presence of t-butyl hydroperoxide, suggesting that this type of redox chemistry may be employed in biology for the control of metal metabolism. Moreover, selenium compounds are likely targets for zinc/thiolate coordination centers in vivo, because the reactions are only partially suppressed by excess glutathione. This specificity and the potential to undergo catalytic reactions at low concentrations suggests that zinc release is a significant aspect of the therapeutic antioxidant actions of selenium compounds in antiinflammatory and anticarcinogenic agents.

Inhibitory sites in enzymes: Zinc removal and reactivation by thionein

Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.

Analysis of masked mutations in familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal-dominant disease characterized by the development of hundreds of adenomatous polyps of the colorectum. Approximately 80% of FAP patients can be shown to have truncating mutations of the APC gene. To determine the cause of FAP in the other 20% of patients, MAMA (monoallelic mutation analysis) was used to independently examine the status of each of the two APC alleles. Seven of nine patients analyzed were found to have significantly reduced expression from one of their two alleles whereas two patients were found to have full-length expression from both alleles. We conclude that more than 95% of patients with FAP have inactivating mutations in APC and that a combination of MAMA and standard genetic tests will identify APC abnormalities in the vast majority of such patients. That no APC expression from the mutant allele is found in some FAP patients argues strongly against the requirement for dominant negative effects of APC mutations. The results also suggest that there may be at least one additional gene, besides APC, that can give rise to FAP.

Zinc metallothionein imported into liver mitochondria modulates respiration

Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiological, micromolar concentrations of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal β-domain of MT; in this system, the isolated C-terminal α-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated β-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apoform of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are critical for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apocytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homology to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biological modulating activity of MT in cellular respiration and energy metabolism in a tissue-specific manner.

Genetic disruption of PPARδ decreases the tumorigenicity of human colon cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARδ isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/β-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARδ null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARδ −/− cells exhibited a decreased ability to form tumors compared with PPARδ +/− and wild-type controls. These data suggest that suppression of PPARδ expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

Top-down morphogenesis of colorectal tumors

One of the fundamental tenets of oncology is that tumors arise from stem cells. In the colon, stem cells are thought to reside at the base of crypts. In the early stages of tumorigenesis, however, dysplastic cells are routinely found at the luminal surface of the crypts whereas the cells at the bases of these same crypts appear morphologically normal. To understand this discrepancy, we evaluated the molecular characteristics of cells isolated from the bases and orifices of the same crypts in small colorectal adenomas. We found that the dysplastic cells at the tops of the crypts often exhibited genetic alterations of adenomatous polyposis coli (APC) and neoplasia-associated patterns of gene expression. In contrast, cells located at the base of these same crypts did not contain such alterations and were not clonally related to the contiguous transformed cells above them. These results imply that development of adenomatous polyps proceeds through a top-down mechanism. Genetically altered cells in the superficial portions of the mucosae spread laterally and downward to form new crypts that first connect to preexisting normal crypts and eventually replace them.