Hot spots for molecular genetic alterations in lung cancer.

Lung cancers are a heterogeneous group of tumors broadly classified as small cell or non-small cell lung cancers. In each case, numerous DNA mutations precede tumor formation, resulting in the activation of growth stimulatory genes and the loss of tumor suppressor genes. The known cellular functions of the tumor suppressor genes most commonly affected in lung cancer are reviewed herein, including the retinoblastoma (Rb) gene on chromosome 13q14, the p53 gene on 17p13, and the cyclin-dependent kinase inhibitor (CDKN2) gene on 9p21. The chromosomal locations for other potential tumor suppressor genes are on chromosomes 3p, 9p, and 11p. Candidate genes in these regions include the von Hippel-Lindau (VHL) gene at 3p25, the ubiquitin-activating enzyme homologue (UBE1L at 3p21, the genes for the dinucleoside polyphosphate hydrolase FHIT and receptor protein-tyrosine phosphatase gamma PTPRG at 3p14.2, the genes for tropomyosin beta (TM1) and a talin homologue (talin) at 9p21, and the H-ras gene at 11p15.

Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1.

Molybdopterin (MPT) is not produced by the Escherichia coli mutants chlA1, chlM, or chlN or by the Neurospora crassa mutant nit-1. Extracts of E. coli chlA1 contain an activity, the converting factor, which is functionally defined by its ability to convert a low-molecular-weight precursor present in crude extracts of N. crassa nit-1 into molybdopterin in vitro. In this study, it has been shown that the converting factor consists of two associative proteins (10 and 25 kilodaltons [kDa]) which can be separated by using either anion-exchange or gel filtration chromatography. Neither protein is able to complement extracts of nit-1 by itself. Analysis of chlA Mu insertion mutants has shown that the two proteins are distinct gene products encoded at the chlA locus. Twelve chlA Mu insertion strains which lacked converting factor activity were deficient in one or both of the proteins. Converting factor activity could be generated by mixing extracts from strains having the 25-kDa protein with those having the 10-kDa protein but not those lacking both proteins. Finally, it was shown that the chlM mutant lacks the 10-kDa protein while the chlN mutant, which contains both the 10- and 25-kDa proteins, lacks a function required to activate the 10-kDa protein.

The biosynthesis of molybdopterin in Escherichia coli. Purification and characterization of the converting factor.

Molybdopterin, the universal component of the pterin molybdenum cofactors, contains a dithiolene group serving to bind Mo. Addition of the dithiolene sulfurs to a molybdopterin precursor requires the activity of the converting factor. Active converting factor has been purified from Escherichia coli chlA1 cells and found to have two subunits of mass 10 and 16 kDa. Electrophoresis of the purified converting factor on denaturing polyacrylamide gels revealed the presence of a 27-kDa protein as well. Partial NH2-terminal amino acid sequencing showed that the 27-kDa protein is a covalent complex of the 10- and 16-kDa proteins. The inactive converting factor purified from E. coli chlN contains both subunits, as established by amino acid sequencing of the purified material, but the 10-kDa subunit is inactive. Absence of the 27-kDa complex in chlN preparations showed that the inactive covalent complex is only formed from the active converting factor. Evidence that activation of the small subunit requires the acquisition of sulfur was obtained from the difference in the molecular masses of the 10-kDa proteins isolated from chlA1 and chlN cells and from the differential sensitivities of the chlA1 and chlN converting factors to iodoacetamide.

In vitro synthesis of molybdopterin from precursor Z using purified converting factor. Role of protein-bound sulfur in formation of the dithiolene.

The pterin component of the molybdenum cofactor, termed molybdopterin, is synthesized in Escherichia coli by enzymes encoded at the chl loci. A late step in the biosynthetic pathway, the conversion of a molybdopterin intermediate, precursor Z, to molybdopterin, requires the activity of a two-subunit protein, the converting factor. Precursor Z has many of the features of molybdopterin but lacks the dithiolene function essential for molybdenum ligation. Conversion of precursor Z to molybdopterin is accomplished by transfer of sulfur to produce the dithiolene. The present study describes an in vitro system for molybdopterin biosynthesis comprised of purified precursor Z and purified converting factor. It is established that these components are sufficient to yield molybdopterin, identified by conversion to its characteristic products, Form A, Form B, and dicarboxamidomethylmolybdopterin. Under conditions of precursor excess, the formation of molybdopterin was stoichiometric with converting factor, as would be expected in the absence of a sulfur-regenerating system. The labile product of the reaction, molybdopterin, remained associated with the converting factor large subunit. These results establish that the source of sulfur for molybdopterin biosynthesis is the converting factor and suggest that in vivo a novel sulfur cycle must function to resupply sulfur to the converting factor.

SSA/RO52gene and expressed sequence tags in an 85 kb region of chromosome segment 11p15.5.

Frequent allelic loss in lung cancer has been described in a region on chromosome segment 11p15.5 (LOH11A). The region is approximately 650 kb in size and flanked by the markers D11S988 centromeric and D11S860 telomeric. Clinical and cell biological studies suggest that it contains a gene associated with metastatic tumor spread. One of the genes identified within this region is SSA/Ro52, which has a RING finger domain and may be involved in gene regulation. We studied this gene for mutations using SSCP analysis and for expression using RT-PCR and Western blotting on lung cancer cell lines and tumor-normal tissue pairs. No mutations and no differences in mRNA or protein expression between tumor tissue and normal tissue pairs were identified. We discovered a novel polymorphic site (SSA44C/T) within exon 1 of this gene. Among 141 primary lung cancers, allelic loss was observed in 16% of informative cases. Our analyses excluded SSA/Ro52 as a tumor-suppressor gene in lung cancer and newly defined the centromeric border of the LOH11A region from D11S988 previously to SSA44C/T. This reduced the region of the putative suppressor gene to 460 to 485 kb. A significant difference (p = 0.01) in the frequency of alleles for this polymorphism between Caucasians and African-Americans was observed. The "T" allele frequency was 0.12 in Caucasians and 0.23 in African-Americans. A genomic EcoRI map over 85 kb surrounding the SSA/Ro52 gene was constructed, and 4 expressed sequence tags were identified by sequencing and studied.

Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts.

Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein. To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright's stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads. We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process. In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice. However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45-60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells. Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.

A 1.4-Mb high-resolution physical map and contig of chromosome segment 11p15.5 and genes in the LOH11A metastasis suppressor region.

The centromeric part of chromosome segment 11p15.5 contains a region of frequent allele loss in many adult solid malignancies. This region, called LOH11A, is lost in 75% of lung cancers and is thought to contain a gene that may function as a metastasis suppressor. Genetic complementation studies have shown suppression of the malignant phenotype including reduction of metastasis formation. We constructed a high-resolution physical map and contig over 1.4 Mb that includes the beta-hemoglobin gene cluster and the gene for the large subunit of ribonucleotide reductase (RRM1). Through sequencing and computerized analysis, we determined that this region contains an unusually large number of transposable elements, which suggests that double-stranded DNA breaks occur frequently here. Twenty-two putative genes were identified. Because of its location at the site of maximal allele loss in the 650-kb LOH11A region and previous functional studies, RRM1 is the most likely candidate gene with metastasis suppressor function. The malignant phenotype, in this case, results from a relative loss of function rather than a complete loss.

Mechanisms of MARCKS gene activation during Xenopus development.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a high affinity cellular substrate for protein kinase C. The MARCKS gene is under multiple modes of transcriptional control, including cytokine- and transformation-dependent, cell-specific, and developmental regulation. This study evaluated the transcriptional control of MARCKS gene expression during early development of Xenopus laevis. Xenopus MARCKS was highly conserved with its mammalian and avian homologues; its mRNA and protein were abundant in the maternal pool and increased after the mid-blastula transition (MBT). The Xenopus MARCKS gene was similar to those of other species, except that a second intron interrupted the 5'- untranslated region. By transiently transfecting XTC-2 cells and microinjecting Xenopus embryos with reporter gene constructs containing serial deletions of 5'-flanking MARCKS sequences, we identified a 124-base pair minimal promoter that was critical for promoter activity. Developmental gel shift assays revealed that a CBF/NF-Y/CP-1-like factor and an Sp1-like factor bound to this region in a manner correlating with the onset of Xenopus MARCKS transcription at MBT. Mutations in the promoter that abolished binding of these two factors also completely inhibited transcriptional activation of the MARCKS gene at MBT. The binding sites for these two factors are highly conserved in the human and mouse MARCKS promoters, suggesting that these elements might also regulate MARCKS transcription in other species. These studies not only increase our knowledge of the transcriptional regulation of the MARCKS genes but also have implications for the mechanisms responsible for zygotic activation of the Xenopus genome at MBT.

Lung cancer and the human gene for ribonucleotide reductase subunit M1 (RRM1).

LOH11A is a region of Chromosome (Chr) 11p15.5 where 75% of lung cancers show loss of heterozygosity (LOH). Clinical and cell biological studies suggest that LOH11A contains a tumor/metastasis suppressor gene. We have mapped this region (650 kb) using overlapping genomic P1/PAC/BAC clones, and one of the genes that we have identified is RRM1. This gene encodes the large subunit (M1) of ribonucleotide reductase, the heterodimeric enzyme that catalyzes the rate-limiting step in deoxyribonucleotide synthesis. By comparing our genomic sequences with the previously published cDNA, we have found that the human gene is composed of 19 exons. It is oriented telomere to centromere and is Alu rich. In order to verify that RRM1 maps within the boundaries of LOH11A, we assessed the frequency of LOH at a SacI polymorphism within intron IX of the gene. We observed LOH in 48% (15/31) of informative lung tumor specimens. To determine whether RRM1 was mutated in tumors, SSCP analysis of the 19 RRM1 exons was performed. No mutations were revealed in 12 pairs of normal and tumor DNA samples. Immunoblots on protein extracts from normal/tumor pairs indicated that a protein of the expected size was present in both. Our conclusion is that RRM1 lies within the LOH11A region, but that its exons are not mutated in tumors. The potential for RRM1 to act as a tumor suppressor is discussed.

Early biochemical events in insulin-stimulated fluid phase endocytosis.

We examined the initial molecular mechanisms by which cells nonselectively internalize extracellular solutes in response to insulin. Insulin-stimulated fluid phase endocytosis (FPE) was examined in responsive cells, and the roles of the insulin receptor, insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3'-kinase (PI 3'-kinase), Ras, and mitogen-activated protein kinase kinase (MEK) were assessed. Active insulin receptors were essential, as demonstrated by the stimulation of FPE by insulin in HIRc-B cells (Rat-1 cells expressing 1.2 x 10(6) normal insulin receptors/cell) but not in untransfected Rat-1 cells or in Rat-1 cells expressing the inactive A/K1018 receptor. IRS-1 expression augmented insulin-stimulated FPE, as assessed in 32D cells, a hematopoietic precursor cell line lacking endogenous IRS-1. Insulin-stimulated FPE was inhibited in mouse brown adipose tissue (BAT) cells expressing the 17N dominant negative mutant Ras and was augmented in cells expressing wild-type Ras. The MEK inhibitor PD-98059 had little effect on insulin-stimulated FPE in BAT cells. In 32D cells, but not in HIRc-B and BAT cells, insulin-stimulated FPE was inhibited by 10 nM wortmannin, an inhibitor of PI 3'-kinase. The results indicate that the insulin receptor, IRS-1, Ras, and, perhaps in certain cell types, PI 3'-kinase are involved in mediating insulin-stimulated FPE.