Cynthia L. Jackson

Cloning of a cDNA encoding a putative human very low density lipoprotein/apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23

We report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. Our results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

Pattern of mutant p53 expression in human astrocytomas suggests the existence of alternate pathways of tumorigenesis

Background. Clinical observations suggest that malignant astrocytomas may arise from well‐differentiated, low‐grade tumors that have undergone anaplastic progression or may develop de novo. Mutations that alter the function of the p53 gene product are thought to play a critical role in astrocytoma tumorigenesis. The authors studied the pattern of mutant p53 expression in astrocytomas to define its role in the formation of malignant tumors by these different pathways.

Monochromosomal rodent-human hybrids from microcell fusion of human lymphoblastoid cells containing an inserted dominant selectable marker

An improved system for the production of a series of rodent-human hybrids selectively retaining single human chromosomes marked in known locations is described. Such hybrids have significant applications in gene mapping and other genetic studies. Human lymphoblastoid lines were infected with the retroviral vector SP-1, which contains the bacterial his-D gene allowing mammalian cells to grow in the presence of histidinol. Microcell fusion of the infected lymphoblastoid cells with CHO cells was used to produce hybrids containing single human chromosomes retained by histidinol selection. Hybrids containing a single human chromosome 9 and a single human chromosome 19 are described. These have been characterized cytogenetically by G-banding, in situ hybridization, and Southern blot analysis.

Postmortem RNA and protein stability in perinatal human lungs.

The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA later (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN2). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA later yielded more total RNA and protein than LN2 freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase–polymerase chain reaction analysis of representative genes (&bgr;-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA later-treated tissues was equivalent to that of LN2-frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA later is equivalent, if not superior, to LN2 for preservation of postmortem RNA and protein in developing human lungs.

Physical and transcriptional map of the hereditary inclusion body myopathy locus on chromosome 9p12-p13.

Hereditary inclusion body myopathy (HIBM) is a group of neuromuscular disorders characterised by adult-onset, slowly progressive distal and proximal muscle weakness and typical muscle pathology. Previously, we have mapped the gene responsible for a recessive form of HIBM to chromosome 9p1 and narrowed the interval to one single YAC clone of 1 Mb in size. As a further step towards the identification of the HIBM gene, we have constructed a detailed physical and transcriptional map of this region. A high resolution BAC contig that includes the HIBM critical region, flanked by marker 327GT4 and D9S1859, was constructed. This contig allowed the precise localisation of 25 genes and ESTs to the proximal region of chromosome 9. The expression pattern of those mapped genes and ESTs was established by Northern blot analysis. In the process of refining the HIBM interval, 13 new polymorphic markers were identified, of which 11 are CA-repeats, and two are single nucleotide polymorphisms. Certainly, this map provides an important integration of physical and transcriptional information corresponding to chromosome 9p12-p13, which is expected to facilitate the cloning and identification not only of the HIBM gene, but also other disease genes which map to this region.

C-type natriuretic peptide expression and pulmonary vasodilation in hypoxia-adapted rats

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are potent pulmonary vasodilators that are upregulated in hypoxia-adapted rats and may protect against hypoxic pulmonary hypertension. To test the hypothesis that C-type natriuretic peptide (CNP) also modulates pulmonary vascular responses to hypoxia, we compared the vasodilator effect of CNP with that of ANP on pulmonary arterial rings, thoracic aortic rings, and isolated perfused lungs obtained from normoxic and hypoxia-adapted rats. We also measured CNP and ANP levels in heart, lung, brain, and plasma in normoxic and hypoxia-adapted rats. Steady-state CNP mRNA levels were quantified in the same organs by relative RT-PCR. CNP was a less potent vasodilator than ANP in preconstricted thoracic aortic and pulmonary arterial rings and in isolated lungs from normoxic and hypoxia-adapted rats. Chronic hypoxia increased plasma CNP (15 ± 2 vs. 6 ± 1 pg/ml; P < 0.05) and decreased CNP in the right atrium (35 ± 14 vs. 65 ± 17 pg/mg protein; P < 0.05) and in the lung (3 ± 1 vs. 14 ± 3 pg/mg protein; P < 0.05) but had no effect on CNP in brain or right ventricle. Chronic hypoxia increased ANP levels fivefold in the right ventricle (49 ± 5 vs. 11 ± 2 pg/mg protein; P < 0.05) but had no effect on ANP in lung or brain. There was a trend toward decreased ANP levels in the right atrium (2,009 ± 323 vs. 2,934 ± 397 pg/mg protein; P = not significant). No differences in CNP transcript levels were observed between the two groups of rats except that the right atrial CNP mRNA levels were lower in hypoxia-adapted rats. We conclude that CNP is a less potent pulmonary vasodilator than ANP in normoxic and hypoxia-adapted rats and that hypoxia raises circulating CNP levels without increasing cardiopulmonary CNP expression. These findings suggest that CNP may be less important than ANP or BNP in protecting against hypoxic pulmonary hypertension in rats.Atrial and brain natriuretic peptides (ANP and BNP, respectively) are potent pulmonary vasodilators that are upregulated in hypoxia-adapted rats and may protect against hypoxic pulmonary hypertension. To test the hypothesis that C-type natriuretic peptide (CNP) also modulates pulmonary vascular responses to hypoxia, we compared the vasodilator effect of CNP with that of ANP on pulmonary arterial rings, thoracic aortic rings, and isolated perfused lungs obtained from normoxic and hypoxia-adapted rats. We also measured CNP and ANP levels in heart, lung, brain, and plasma in normoxic and hypoxia-adapted rats. Steady-state CNP mRNA levels were quantified in the same organs by relative RT-PCR. CNP was a less potent vasodilator than ANP in preconstricted thoracic aortic and pulmonary arterial rings and in isolated lungs from normoxic and hypoxia-adapted rats. Chronic hypoxia increased plasma CNP (15 +/- 2 vs. 6 +/- 1 pg/ml; P < 0.05) and decreased CNP in the right atrium (35 +/- 14 vs. 65 +/- 17 pg/mg protein; P < 0.05) and in the lung (3 +/- 1 vs. 14 +/- 3 pg/mg protein; P < 0.05) but had no effect on CNP in brain or right ventricle. Chronic hypoxia increased ANP levels fivefold in the right ventricle (49 +/- 5 vs. 11 +/- 2 pg/mg protein; P < 0.05) but had no effect on ANP in lung or brain. There was a trend toward decreased ANP levels in the right atrium (2,009 +/- 323 vs. 2,934 +/- 397 pg/mg protein; P = not significant). No differences in CNP transcript levels were observed between the two groups of rats except that the right atrial CNP mRNA levels were lower in hypoxia-adapted rats. We conclude that CNP is a less potent pulmonary vasodilator than ANP in normoxic and hypoxia-adapted rats and that hypoxia raises circulating CNP levels without increasing cardiopulmonary CNP expression. These findings suggest that CNP may be less important than ANP or BNP in protecting against hypoxic pulmonary hypertension in rats.

Oncogenic ALK Fusion in Rare and Aggressive Subtype of Colorectal Adenocarcinoma as a Potential Therapeutic Target

Purpose: Chromosomal translocations in the anaplastic lymphoma kinase (ALK) gene have been identified as oncogenic drivers in lung adenocarcinomas and other tumors, recently including rare cases of colorectal carcinoma. We identified a patient with refractory metastatic colorectal carcinoma harboring a STRN–ALK gene fusion who achieved an exceptional clinical benefit to the ALK inhibitor ceritinib. Our goal was to further define the clinicopathologic features of ALK-rearranged colorectal carcinoma in a large cohort. Experimental Design: Clinical cases of colorectal carcinoma evaluated by comprehensive genomic profiling (CGP) or by ALK immunohistochemistry (IHC) were reviewed retrospectively. FISH and microsatellite instability (MSI) analyses were performed. Results: Nine colorectal carcinoma cases harbored ALK gene fusions. Six cases were identified by CGP of 3,157 colorectal carcinoma (0.2%) and three by IHC of 2,980 colorectal carcinoma (0.1%). The ALK fusions involved known ALK partners EML4, C2orf44, CAD, and the novel STRN, PPP1R21, SENPF, MAPRE3, and PRKAP1B partners. These advanced-stage colorectal carcinomas lacked mutations in other oncogenic drivers, predominantly involved the proximal colon, and often exhibited MSI and mucinous phenotype. The index patient was treated with the ALK inhibitor ceritinib, resulting in a marked decrease in size of a skin metastasis, and resolution by computerized tomography of all contrast enhancing tumor. After 9 months of treatment, biopsy of progressive disease demonstrated a KRAS mutation, consistent with acquired resistance to ceritinib. Conclusions: Colorectal carcinoma harboring ALK fusions represent a rare aggressive subtype of colorectal carcinoma with distinct clinicopathologic features. This report provides the first clinical evidence that such patients may benefit from targeted monotherapy with ALK inhibitors. Clin Cancer Res; 22(15); 3831–40. ©2016 AACR.

Subcellular accumulation of β-carotene and retinoids in growth-inhibited NCI-H69 small cell lung cancer cells

Delivery of β-carotene in tetrahydrofuran slowed the growth of NCI-H69 small cell lung cancer cells. Analysis of cells and cellular fractions revealed that β-carotene-treated cells accumulated β-carotene as well as some polar metabolites, primarily in the crude nuclei. Cells were grown at 1 × 105 cells/ml and treated with 20 μM β-carotene. Growth monitoring up to 15 days indicated an inverse relationship between the duration of β-carotene treatment and the rate of cell growth. Reverse-phase high-performance liquid chromatography analysis of treated cells showed the presence of β-carotene, retinoic acid, retinol, and retinal, with β-carotene accounting for the major material recovered. When cellular fractions were analyzed for β-carotene, it was found to be located primarily in the crude nuclei. These results demonstrate that treatment of small cell lung cancer cells with β-carotene results in a reduced growth of the cells. Further investigation is required to show a direct effect of β-carotene or its intr...

Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study.

ObjectivesK-ras gene product in the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway is critical in the development of certain types of malignancies. K-ras mutation–associated pancreatic and ovarian carcinomas often display mucinous differentiation. Previous studies have shown that k-ras mutation is found in 10% to 30% of endometrial carcinomas. We investigated k-ras mutations in several morphologic subtypes of endometrial carcinomas with particular emphasis on various degrees of mucinous differentiation. MethodsGenomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections. Polymerase chain reaction amplification for k-ras codons 12 and 13 were performed, followed by sequencing using capillary electrophoresis. The Fisher exact test is used to compare the prevalent difference of k-ras mutation among the groups. P < 0.05 was considered significant. ResultsK-ras mutations were detected in 8 (80%) of 10 mucinous carcinomas, 12 (67%) of 18 endometrioid carcinomas (ECs) with significant mucinous differentiation (ECMD), 4 (25%) of 16 ECs, and 1 (9%) of 11 serous carcinomas. The differences were statistically significant between mucinous carcinomas versus EC (P < 0.01) and ECMD versus EC (P < 0.05). ConclusionThe findings suggest that mucinous carcinoma and endometrioid carcinoma with significant mucinous component are more likely to be associated with k-ras mutation. Potential clinical implications of k-ras mutation lies in the management of recurrent or higher-stage endometrial mucinous tumors, which would not be responsive to treatment protocols containing epidermal growth factor receptor inhibitors.Objectives K-ras gene product in the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway is critical in the development of certain types of malignancies. K-ras mutation–associated pancreatic and ovarian carcinomas often display mucinous differentiation. Previous studies have shown that k-ras mutation is found in 10% to 30% of endometrial carcinomas. We investigated k-ras mutations in several morphologic subtypes of endometrial carcinomas with particular emphasis on various degrees of mucinous differentiation. Methods Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections. Polymerase chain reaction amplification for k-ras codons 12 and 13 were performed, followed by sequencing using capillary electrophoresis. The Fisher exact test is used to compare the prevalent difference of k-ras mutation among the groups. P < 0.05 was considered significant. Results K-ras mutations were detected in 8 (80%) of 10 mucinous carcinomas, 12 (67%) of 18 endometrioid carcinomas (ECs) with significant mucinous differentiation (ECMD), 4 (25%) of 16 ECs, and 1 (9%) of 11 serous carcinomas. The differences were statistically significant between mucinous carcinomas versus EC (P < 0.01) and ECMD versus EC (P < 0.05). Conclusion The findings suggest that mucinous carcinoma and endometrioid carcinoma with significant mucinous component are more likely to be associated with k-ras mutation. Potential clinical implications of k-ras mutation lies in the management of recurrent or higher-stage endometrial mucinous tumors, which would not be responsive to treatment protocols containing epidermal growth factor receptor inhibitors.

Construction and characterization of radiation hybrids for chromosome 9, and their use in mapping cosmid probes on the chromosome

Radiation hybrids were produced from a monochromosomal microcell hybrid (PK87-9) which contains only human chromosome 9 with an inserted marker on 9p. Doses of radiation ranging from 1000 to 8000 rads were used to produce a series of hybrids with different size fragments of human chromosome 9. The inserted dominant selectable marker was used to select for hybrids that preferentially maintain fragments of 9p. A panel of 53 radiation hybrids were characterized for 17 chromosome 9 markers. In addition, 17 hybrids were analyzed by fluorescent in situ hybridization (FISH). Hybrids were produced with breaks on both 9p and 9q, many of which appear to contain a single fragment of human chromosome 9. These hybrid cell lines were used to regionally localize 31 cosmids isolated from a chromosome 9 cosmid library. Six cosmids were mapped to intervals on 9p, six cosmids mapped to the centromeric region of the chromosome, and 19 mapped to 9q.