Biomedical subjects

M C Willingham

Publications and source records attributed to M C Willingham.

At least 19 recordsLinked to original sources

PubMed2006

Expression of Dmp1 in specific differentiated, nonproliferating cells and its regulation by E2Fs.

Dmp1 is a Myb-like transcription factor that transmits oncogenic Ras-Raf signaling to the Arf-p53 pathway and induces cell cycle arrest. Immunohistochemical staining was performed to identify the pattern of Dmp1 expression in normal murine tissues compared with the proliferation marker, Ki67. In thymus, the nuclei of mature T lymphocytes in the medulla were strongly positive for Dmp1, whereas Ki67 was detected only in the cortex. In intestine, Dmp1 was detected in the nuclei of superficial layers of the villi, whereas Ki67-positive cells were confined to the lower one-third of the crypt. Double staining for Dmp1 and Ki67 revealed that these two proteins were expressed in mutually exclusive fashion in nearly all the tissues examined. Subsets of E2Fs were specifically bound to the Dmp1 promoter upon mitogenic signaling and E2Fs 1-4 inhibited the Dmp1 promoter in a reporter assay. The Dmp1 promoter was repressed when the cells entered the S to G2/M phase of the cell cycle when both Dmp1 and Arf expressions were downregulated. The Dmp1 mRNA was not downregulated by serum in E2F-DB(+) cells, suggesting that the Dmp1 promoter repression is E2F-dependent. This explains why the Dmp1 and Ki67-positive cells are stained in mutually exclusive fashion in normal tissues.

3T3 Cells↗

PubMed2006

PPARgamma insufficiency promotes follicular thyroid carcinogenesis via activation of the nuclear factor-kappaB signaling pathway.

The molecular genetic events underlying thyroid carcinogenesis are poorly understood. Mice harboring a knock-in dominantly negative mutant thyroid hormone receptor beta (TRbetaPV/PV mouse) spontaneously develop follicular thyroid carcinoma similar to human thyroid cancer. Using this mutant mouse, we tested the hypothesis that the peroxisome proliferator-activated receptor gamma (PPARgamma) could function as a tumor suppressor in thyroid cancer in vivo. Using the offspring from the cross of TRbetaPV/+ and PPARgamma+/- mice, we found that thyroid carcinogenesis progressed significantly faster in TRbetaPV/PV mice with PPARgamma insufficiency from increased cell proliferation and reduced apoptosis. Reduced PPARgamma protein abundance led to the activation of the nuclear factor-kappaB signaling pathway, resulting in the activation of cyclin D1 and repression of critical genes involved in apoptosis. Treatment of TRbetaPV/PV mice with a PPARgamma agonist, rosiglitazone, delayed the progression of thyroid carcinogenesis by decreasing cell proliferation and activation of apoptosis. These results suggest that PPARgamma is a critical modifier in thyroid carcinogenesis and could be tested as a therapeutic target in thyroid follicular carcinoma.

Animals↗

PubMed2003

Marked potentiation of the dominant negative action of a mutant thyroid hormone receptor beta in mice by the ablation of one wild-type beta allele.

Mutations in the thyroid hormone receptor (TR) beta gene result in resistance to thyroid hormone (RTH), characterized by reduced sensitivity of tissues to thyroid hormone. To understand which physiological TR pathways are affected by mutant receptors, we crossed mice with a dominantly negative TRbeta mutation (TRbetaPV) with mice carrying a TRbeta null mutation (TRbeta(-/-)) to determine the consequences of the TRbetaPV mutation in the absence of wild-type TRbeta. TRbeta(PV/-) mice are distinct from TRbeta(+/-) mice that did not show abnormalities in thyroid function tests. TRbeta(PV/-) mice are also distinct from TRbeta(PV/+) and TRbeta(-/-) mice in that the latter shows mild dysfunction in the pituitary-thyroid axis, whereas the former exhibit very severe abnormalities, including extensive papillary hyperplasia of the thyroid epithelium, indistinguishable from that observed in TRbeta(PV/PV) mice. Similar to TRbeta(PV/PV) mice, TRbeta(PV/-) mice exhibited impairment in weight gain. Moreover, the abnormal regulation patterns of T3-target genes in the tissues of TRbeta(PV/-) and TRbeta(PV/PV) mice were strikingly similar. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed with TRalpha1 for binding to thyroid hormone response elements in TRbeta(PV/-) mice as effectively as in TRbeta(PV/PV) mice. Thus, the actions of mutant TRbeta are markedly potentiated by the ablation of the second TRbeta allele, suggesting that interference with wild-type TRalpha1-mediated gene regulation by mutant TRbeta leads to severe RTH.

Alleles↗

PubMed2003

Diphtheria toxin-interleukin-3 fusion protein (DT(388)IL3) prolongs disease-free survival of leukemic immunocompromised mice.

The novel fusion protein DT(388)IL3, composed of the catalytic and translocation domains of diphtheria toxin (DT(388)) fused with a Met-His linker to human interleukin 3 (IL-3), was tested for anti-leukemia efficacy in an in vivo model of differentiated human acute myeloid leukemia (AML). Six-week-old female SCID mice were irradiated with 350 cGy, inoculated 24 h later with 20 million (i.v., i.p., or s.c.) TF1 cells transfected with the v-SRC oncogene, and treated i.p., starting 24 h later, with up to five daily injections of saline, DT(388)IL3 (2 microg), DT(388)GMCSF (2 microg), DAB(389)IL2 (2 microg), or cytarabine (80 microg) or two weekly injections of anti-CD33-calicheamicin conjugate (5 microg). Animals were monitored twice daily, and moribund animals killed and necropsied. Control animals had a median disease-free survival (DFS) of 37 days (i.v., n = 45), 35 days (i.p., n = 20), and 21 days (s.c., n = 20), respectively. Only 5/49 (10%) of the DT(388)IL3 treated i.v. inoculated animals died with leukemia. Median DFS with i.v., i.p. and s.c. tumor inoculated animals was prolonged by fusion protein treatment to >120 days, 66 days and 31 days (P < 0.001, = 0.0003, and = 0.0006), respectively. Median DFS with s.c. tumor inoculated animals was also prolonged by other active anti-leukemia agents (DT(388)GMCSF, cytarabine and anti-CD33-calicheamicin) relative to controls by 67%, 172% and 47% (P < 0.0001, <0.0001, and =0.0004), respectively. In contrast, median DFS with s.c. tumor inoculated animals treated with DAB(389)IL2 non-significantly reduced by 13% relative to controls (P = 0.21). Thus, DT(388)IL3 fusion protein demonstrates in vivo anti-leukemia efficacy and warrants further preclinical development for treatment of chemo-resistant, IL-3 receptor positive AML patients.

Acute Disease↗

PubMed2001

A targeted dominant negative mutation of the thyroid hormone alpha 1 receptor causes increased mortality, infertility, and dwarfism in mice.

Mutations in the thyroid hormone receptor beta (TRbeta) gene result in resistance to thyroid hormone. However, it is unknown whether mutations in the TRalpha gene could lead to a similar disease. To address this question, we prepared mutant mice by targeting mutant thyroid hormone receptor kindred PV (PV) mutation to the TRalpha gene locus by means of homologous recombination (TRalpha1PV mice). The PV mutation was derived from a patient with severe resistance to thyroid hormone that has a frameshift of the C-terminal 14 aa of TRbeta1. We knocked in the same PV mutation to the corresponding TRalpha gene locus to compare the phenotypes of TRalpha1(PV/+) mice with those of TRbeta(PV/+) mice. TRalpha1(PV/+) mice were viable, indicating that the mutation of the TRalpha gene is not embryonic lethal. In drastic contrast to the TRbeta(PV/+) mice, which do not exhibit a growth abnormality, TRalpha1(PV/+) mice were dwarfs. These dwarfs exhibited increased mortality and reduced fertility. In contrast to TRbeta(PV/+) mice, which have a hyperactive thyroid, TRalpha1(PV/+) mice exhibited mild thyroid failure. The in vivo pattern of abnormal regulation of T3 target genes in TRalpha1(PV/+) mice was unique from those of TRbeta(PV/+) mice. The distinct phenotypes exhibited by TRalpha1(PV/+) and TRbeta(PV/+) mice indicate that the in vivo functions of TR mutants are isoform-dependent. The TRalpha1(PV/+) mice may be used as a tool to uncover human diseases associated with mutations in the TRalpha gene and, furthermore, to understand the molecular mechanisms by which TR isoforms exert their biological activities.

Animals↗

PubMed2001

Oncogene-dependent engraftment of human myeloid leukemia cells in immunosuppressed mice.

We have developed an in vivo model of differentiated human acute myeloid leukemia (AML) by retroviral infection of the cytokine-dependent AML cell line TF-1 with the v-Src oncogene. When injected either intravenously or intraperitoneally into 300 cGy irradiated SCID mice, animals formed multiple granulocytic sarcomas involving the adrenals, kidneys, lymph nodes and other organs. The mean survival time was 34+/-10 days (n = 40) after intravenous injection and 24+/-3 days (n = 5) after intraperitoneal injection of 20 million cells. The cells recovered from leukemic animals continued to express interleukin-3 receptors and remained sensitive to the diphtheria fusion protein DT388IL3. Further, these granulocytic sarcoma-derived cells grew again in irradiated SCID mice (n = 10). The cytogenetic abnormalities observed prior to inoculation in mice were stably present after in vivo passage. Similar to the results with v-Src transfected TF-1 cells, in vivo leukemic growth was observed with TF-1 cells transfected with the human granulocyte-macrophage colony-stimulating factor gene (n = 5) and with TF-1 cells recovered from subcutaneous tumors in nude mice (n = 5). In contrast, TF-1 cells expressing v-Ha-Ras (n = 5), BCR-ABL (n = 5), or activated Raf-1 (n = 44) did not grow in irradiated SCID mice. This is a unique, reproducible model for in vivo growth of a differentiated human acute myeloid leukemia and may be useful in the assessment of anti-leukemic therapeutics which have human-specific molecular targets such as the interleukin-3 receptor.

Animals↗

PubMed2001

p53-independent apoptosis mediated by tachpyridine, an anti-cancer iron chelator.

Iron is involved in essential biochemical reactions ranging from respiration to DNA synthesis. Consequently, iron deprivation has been proposed as a strategy for inhibition of tumor cell growth. We recently described a novel iron chelator, tachypyridine [N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane], and demonstrated that it not only inhibited growth of cultured tumor cells, but was actively cytotoxic. Here we explore the mechanisms underlying tachpyridine cytotoxicity. Using several criteria, including time-lapse video microscopy, DNA staining and TUNEL assays, tachpyridine was shown to specifically induce apoptotic cell death. Further, unlike numerous cytotoxic chemotherapeutic drugs which induce apoptosis by activating p53-dependent pathways, tachpyridine-mediated cell death did not require p53 activation. Although immunoblotting revealed rapid accumulation of p53 following treatment with tachpyridine, p21(WAF1) was not induced. Further, neither cytotoxicity nor apoptosis required p53. p53 null human lung cancer H1299 cells transfected with an ecdysone-inducible p53 exhibited equivalent sensitivity to tachpyridine in the presence and absence of p53, demonstrating the lack of requirement for p53 in an isogenic cell system. Further, time-lapse video microscopy and TUNEL assays demonstrated that both p53 null and p53 wild-type cells underwent apoptotic cell death in response to tachpyridine. In addition, in 55 human cancer cell lines the mean GI(50) of tachpyridine in cells with mutant p53 was virtually identical to the GI(50) in cells with wild-type p53. These results demonstrate that tachpyridine initiates an apoptotic mode of cell death that does not require functional p53. Since over 50% of human tumors contain a functionally defective p53 that reduces sensitivity to commonly used chemotherapeutic agents, such as etoposide and cisplatin, the ability of tachpyridine to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy.

Anticarcinogenic Agents↗

PubMed2001

Growth hormone increases regional coronary blood flow and capillary density in aged rats.

In this study, we examined the effects of age and growth hormone replacement on both coronary blood flow and capillary density. Blood flow was measured by using [(14)C]-iodoantipyrine in three groups of anesthetized Brown Norway x Fischer 344 rats: young vehicle-treated animals (6 months; n = 13), old vehicle treated animals (30 months; n = 9), and old animals treated with bovine growth hormone (200 microg/kg) twice a day for 30 days (30 months; n = 7). Capillary density was measured by color segmentation analysis of sections stained for platelet endothelial cell adhesion molecule-1. In all regions examined, coronary blood flow decreased with age, and growth hormone administration resulted in an increase in flow compared to vehicle-treated animals. Capillary density decreased with age in the apex and the left ventricular middle segment. In response to growth hormone administration, capillary density increased significantly in the apex but not in other regions of the heart. Our results demonstrate that growth hormone enhances regional myocardial blood flow in the aged heart and suggest that part of this effect could be due to an increase in capillary density.

Aging↗

PubMed2001

Matrix protein and another viral component contribute to induction of apoptosis in cells infected with vesicular stomatitis virus.

The induction of apoptosis in host cells is a prominent cytopathic effect of vesicular stomatitis virus (VSV) infection. The viral matrix (M) protein is responsible for several important cytopathic effects, including the inhibition of host gene expression and the induction of cell rounding in VSV-infected cells. This raises the question of whether M protein is also involved in the induction of apoptosis. HeLa or BHK cells were transfected with M mRNA to determine whether M protein induces apoptosis when expressed in the absence of other viral components. Expression of M protein induced apoptotic morphological changes and activated caspase-3 in both cell types, indicating that M protein induces apoptosis in the absence of other viral components. An M protein containing a point mutation that renders it defective in the inhibition of host gene expression (M51R mutation) activated little, if any, caspase-3, while a deletion mutant lacking amino acids 4 to 21 that is defective in the virus assembly function but fully functional in the inhibition of host gene expression was as effective as wild-type (wt) M protein in activating caspase-3. To determine whether M protein influences the induction of apoptosis in the context of a virus infection, the M51R M protein mutation was incorporated onto a wt background by using a recombinant infectious cDNA clone (rM51R-M virus). The timing of the induction of apoptosis by rM51R-M virus was compared to that by the corresponding recombinant wt (rwt) virus and to that by tsO82 virus, the mutant virus in which the M51R mutation was originally identified. In HeLa cells, rwt virus induced apoptosis faster than did rM51R-M virus, demonstrating a role for M protein in the induction of apoptosis. In contrast to the results obtained with HeLa cells, rwt virus induced apoptosis more slowly than did rM51R-M virus in BHK cells. This indicates that a viral component other than M protein contributes to induction of apoptosis in BHK cells and that wt M protein acts to delay induction of apoptosis by the other viral component. tsO82 virus induced apoptosis more rapidly than did rM51R-M virus in both HeLa and BHK cells. These two viruses contain the same point mutation in their M proteins, suggesting that sequence differences in genes other than that for M protein affect their rates of induction of apoptosis.

Animals↗

PubMed2001

Growth hormone reverses age-related cardiac myofilament dysfunction in rats.

We tested the hypotheses that aging is associated with a reduction in overall cardiac contractility and myofilament force generation that could be reversed with growth hormone (GH) replacement. Three groups of male Brown-Norway rats were studied: young (Y(SAL): 8 mo old, n = 13), old (O(SAL): 28 mo old, n = 13), and old GH-treated (O(GH): 28 mo old, n = 12; 300 microg bovine GH, twice a day for 30 days). The left ventricular (LV) pressure-volume relation was derived in isolated hearts, after which isolated trabecular muscles from these hearts were permeabilized and maximal myofilament force generation (Fmax) was measured. LV developed pressures at a LV volume of 0.3 ml were significantly depressed with age: 84 +/- 6 vs. 71 +/- 6 mmHg (Y(SAL) vs. O(SAL), respectively, P = 0.001) and not restored by GH (69 +/- 4 mmHg). Fmax was reduced in the aged hearts: 47.5 +/- 3.12 vs. 35.9 +/- 3.03 mN/mm2 (Y(SAL) vs. O(SAL), respectively, P = 0.014) but was restored with GH replacement to 46.7 +/- 3.12 mN/mm2 (O(SAL) vs. O(GH), P = 0.021). Our results suggest that cellular myofilament contractility is reduced with aging and restored with GH replacement.

Aging↗

PubMed2001

Advances in cytochemical methods for detection of apoptosis.

In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic process. We also show that only a minority of cells label with fluorescent annexin V during apoptosis but that apoptotic cells continue to internalize material from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assays for caspase substrate cleavage products, the detection of cleaved cytokeratin 18. It is these newer cleavage product assays that perhaps hold the greatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues. (J Histochem Cytochem 49:821-832, 2001)

Annexin A5↗

PubMed2000

Mice with a targeted mutation in the thyroid hormone beta receptor gene exhibit impaired growth and resistance to thyroid hormone.

Patients with mutations in the thyroid hormone receptor beta (TRbeta) gene manifest resistance to thyroid hormone (RTH), resulting in a constellation of variable phenotypic abnormalities. To understand the molecular basis underlying the action of mutant TRbeta in vivo, we generated mice with a targeted mutation in the TRbeta gene (TRbetaPV; PV, mutant thyroid hormone receptor kindred PV) by using homologous recombination and the Cre/loxP system. Mice expressing a single PV allele showed the typical abnormalities of thyroid function found in heterozygous humans with RTH. Homozygous PV mice exhibit severe dysfunction of the pituitary-thyroid axis, impaired weight gains, and abnormal bone development. This phenotype is distinct from that seen in mice with a null mutation in the TRbeta gene. Importantly, we identified abnormal expression patterns of several genes in tissues of TRbetaPV mice, demonstrating the interference of the mutant TR with the gene regulatory functions of the wild-type TR in vivo. These results show that the actions of mutant and wild-type TRbeta in vivo are distinct. This model allows further study of the molecular action of mutant TR in vivo, which could lead to better treatment for RTH patients.

Alleles↗

PubMed2000

Expression, transport properties, and chromosomal location of organic anion transporter subtype 3.

The rat and mouse organic anion-transporting polypeptides (oatp) subtype 3 (oatp3) were cloned to further define components of the intestinal bile acid transport system. In transfected COS cells, oatp3 mediated Na(+)-independent, DIDS-inhibited taurocholate uptake (Michaelis-Menten constant approximately 30 microM). The oatp3-mediated uptake rates and affinities were highest for glycine-conjugated dihydroxy bile acids. In stably transfected, polarized Madin-Darby canine kidney (MDCK) cells, oatp3 mediated only apical uptake of taurocholate. RT-PCR analysis revealed that rat oatp3, but not oatp1 or oatp2, was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. An antibody directed to the carboxy terminus localized oatp3 to the apical brush-border membrane of rat jejunal enterocytes. The mouse oatp3 gene was localized to a region of mouse chromosome 6. This region is syntenic with human chromosome 12p12, where the human OATP-A gene was mapped, suggesting that rodent oatp3 is orthologous to the human OATP-A. These transport and expression properties suggest that rat oatp3 mediates the anion exchange-driven absorption of bile acids previously described for the proximal small intestine.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

The pathogenesis of foam cell formation: modified LDL stimulates uptake of co-incubated LDL via macropinocytosis.

Previously, modified LDLs were shown to stimulate macropinocytosis in pigeon macrophages. Simultaneous intracellular trafficking of LDL and AcLDL, differentially labeled with colloidal gold, was done to determine whether uptake of LDL, which does not cause foam cell formation, was internalized via a separate route from AcLDL, which stimulates foam cell formation. AcLDL and LDL were followed at either low (12 microg/mL) concentrations near the saturation of high affinity binding sites or high (50 to 150 microg/mL) lipoprotein concentrations used to induce foam cell formation. The colloidal gold distribution and percentage of co-labeling as observed by transmission electron microscopy were determined for organelles involved with coated-pit endocytosis or macropinocytosis. LDL simultaneously incubated with AcLDL on macrophages at the low concentration was predominately internalized via coated-pit endocytosis. AcLDL was internalized via both coated-pit endocytosis and macropinocytosis at low concentration. At higher lipoprotein concentrations (50 to 150 microg/mL), AcLDL continued to be internalized via macropinocytosis. Interestingly, a significant portion of the co-incubated LDL, at high concentrations, also trafficked via macropinocytosis. LDL internalized by macropinosomes at high lipoprotein concentrations suggests that AcLDL-stimulated macropinocytosis might increase uptake of co-incubated lipoproteins. When (125)I-LDL was incubated with cold AcLDL, LDL degradation at 37 degrees C doubled, without a corresponding increase in cell association or total binding of LDL at 4 degrees C. These studies suggest that modified LDL-stimulated macropinocytosis is a mechanism for increased degradation of co-incubated LDL potentially leading to foam cell formation.

Animals↗

PubMed2000

Cell-specific expression of group X and group V secretory phospholipases A(2) in human lung airway epithelial cells.

Secretory phospholipase A(2) (sPLA(2)) enzymes contribute to inflammatory injury in human lungs by several mechanisms, including eicosanoid production and hydrolytic damage to surfactant phospholipids. Several distinct sPLA(2) genes have been described in human tissue but little is known regarding their presence, localization, or function(s) within lungs. We hypothesized that sPLA(2)s would have cell-specific distributions within lung. We used reverse transcriptase/polymerase chain reaction to identify sPLA(2) messenger RNAs (mRNAs) in adult human lung tissue. Resulting complementary DNA (cDNA) sequences indicated that total lung extracts contained mRNA for Groups IB, IIA, V, and X sPLA(2). An epithelial cell line, BEAS cells, expressed only Groups IIA, V, and X. We used these cDNAs to clone these enzymes, especially the recently described Group X and Group V enzymes. Digoxigenin-labeled complementary RNA probes were used to determine localization of each sPLA(2) by in situ hybridization of human lung. Hybridization was strongly positive for Group X and Group V in airway epithelial cells, which failed to hybridize Group IB or IIA probes. Although four known mammalian sPLA(2) isotypes were expressed in lung, only Group X and Group V sPLA(2) mRNAs appear uniquely expressed in airway epithelium, suggesting they could provide a mechanism of pulmonary surfactant hydrolysis during lung injury.

Animals↗

PubMed2000

Imaging and phase I study of 111In- and 90Y-labeled anti-LewisY monoclonal antibody B3.

B3 is a murine monoclonal antibody (mAb) that recognizes a LewisY carbohydrate antigen present on the surface of many carcinomas. An imaging and Phase I trial was performed to study the ability of 111In-mAb B3 to image known metastasis and determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), kinetics, and biodistribution of 90Y-mAb B3. Patients (n = 26) with advanced epithelial tumors that express the LewisY antigen were entered. All patients received 5 mCi of 111In-mAb B3 for imaging. 90Y-mAb B3 doses were escalated from 5 to 25 mCi in 5-mCi increments. 111In-mAb B3 and 90Y-mAb B3 were coadministered over a 1-h infusion. Definite tumor imaging was observed in 20 of 26 patients. Sites imaged included lung, liver, bone, and soft tissues. The MTD of 90Y-mAb B3 was determined to be 20 mCi. The DLTs were neutropenia and thrombocytopenia. Tumor doses ranged from 7.7 to 65.1 rad/mCi. 111In- and 90Y-mAb B3 serum pharmacokinetics (n = 23) were found to be similar. The amount of B3 administered (5, 10, and 50 mg) did not alter the pharmacokinetics. Bone marrow biopsies (n = 23) showed 0.0038+/-0.0016% of injected dose/gram for 111In-mAb B3 compared to 0.0046+/-0.0017% of injected dose/gram for 90Y-mAb B3 (P = 0.009). When given to patients with carcinomas that express the LewisY antigen, 111In-mAb B3 demonstrated good tumor localization. The MTD of 90Y-mAb B3 is 20 mCi, with myelosuppression as the DLT. Higher doses of radioactivity need to be delivered to achieve an antitumor effect. Humanized mAb B3 is being developed for evaluation in radioimmunotherapy. A clinical trial to explore the use of higher doses of 90Y-mAb B3 with autologous stem cell support is planned.

Adult↗

PubMed2000

Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.

Two closely related enzymes with more than 50% sequence identity have been identified that catalyze the esterification of cholesterol using acyl-CoA substrates, namely acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2. Both are membrane-spanning proteins believed to reside in the endoplasmic reticulum of cells. ACAT2 has been hypothesized to be associated with lipoprotein particle secretion whereas ACAT1 is ubiquitous and may serve a more general role in cellular cholesterol homeostasis. We have prepared and affinity purified rabbit polyclonal antibodies unique to either ACAT enzyme to identify their cellular localization in liver and intestine, the two main lipoprotein-secreting tissues of the body, and for comparison, kidney and adrenal. In the liver, ACAT2 was identified in the rough endoplasmic reticulum of essentially all hepatocytes whereas ACAT1 was confined to cells lining the intercellular spaces among hepatocytes in a pattern typical of Kupffer cells. In the intestine, ACAT2 signal was strongly present in the apical third of the mucosal cells, whereas ACAT1 staining was diffuse throughout the mucosal cell, but with strong signal in goblet cells, Paneth cells, and villus macrophages. In the kidney, ACAT1 immunostaining was specific for the distal tubules and podocytes within the glomerulus. In the adrenal, ACAT1 signal was strongly present in the cells of the cortex, and absent from other adrenal cell types. No ACAT2 signal was identified in the kidney or adrenal. We conclude that only the cells of the liver and intestine that secrete apolipoprotein B-containing lipoproteins contain ACAT2, whereas ACAT1 is present in numerous other cell types. The data clearly suggest separate functions for these two closely related enzymes, with ACAT2 being most closely associated with plasma cholesterol levels.

Adrenal Glands↗

PubMed1999

Influence of coenzyme A-independent transacylase and cyclooxygenase inhibitors on the proliferation of breast cancer cells.

Recent studies have demonstrated that arachidonic acid (AA) may serve as an important signal that blocks cell proliferation of certain neoplastic cells. The current study was conducted to determine whether disruption of AA homeostasis influences breast cancer cell proliferation and death. Initial experiments revealed that inhibition of AA remodeling through membrane phospholipids by inhibitors of the enzyme, coenzyme A-independent transacylase (CoA-IT), attenuates the proliferation of the estrogen receptor-negative, MDA-MB-231, and estrogen receptor-positive, MCF-7 breast cancer cell lines. This growth inhibition was accompanied by a marked accumulation of AA in both free fatty acid and triglyceride forms, a marker of intracellular AA stress within mammalian cells. Cell cycle synchronization experiments revealed that the CoA-IT inhibitor, SB-98625, blocked MDA-MB-231 cell replication in early to mid G1 phase. Time-lapse video microscopy, used to observe the changes in cell morphology associated with apoptosis, indicated that SB-98625 treatment induced early rounding and occasional blebbing but not late apoptotic events, blistering, and lysis. The cyclooxygenase inhibitors, NS-398 and indomethacin, were found to be less potent blockers of cell proliferation and poor inducers of cellular AA accumulation than CoA-IT inhibitors in these breast cancer cell lines. Finally, AA provided exogenously blocked the proliferation of MCF-7 cells, and this effect could be attenuated in MCF-7 cells overexpressing the glutathione peroxidase gene, GSHPx-1. Taken together, these experiments suggest that disruption of AA remodeling in a manner that increases intracellular AA may represent a novel therapeutic strategy to reduce cancer cell proliferation and that an oxidized AA metabolite is likely to mediate this effect.

Acyltransferases↗