Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.cancer mutation ͉ oncogene addiction ͉ pharmacogenomic ͉ targeted therapies ͉ tumor progression model T he construction of model systems that accurately recapitulate the genetic alterations present in human cancer is a prerequisite to understand the cellular properties imparted by the mutated alleles and to identify genotype and tumor-specific pharmacological responses. In this regard, mammalian cell lines have been widely used as model systems to functionally characterize cancer alleles carrying point mutations and to develop and validate anticancer drugs. These models typically involve the ectopic expression (by means of plasmid transfection or viral infection) of mutated cDNAs in human or mouse cells (1). Although these approaches have yielded remarkable results, they are typically hampered by at least two caveats. First, the expression is achieved by transient or stable transfection of cDNAs, often resulting in over-expression of the target allele at levels that do not recapitulate what occurs in human cancers. Second, the expression of the mutated cDNA is achieved under the control of nonendogenous viral promoters. As a result, the mutated alleles cannot be appropriately (endogenously) modulated in the target cells. While such systems in which mutated oncogenes are ectopically expressed under exogenous promoters have been instrumental in dissecting their oncogenic properties, they have also led to controversial results. For example, studies focused on oncogene-mediated transformation and senescence have generated conflicting data depending on whether the cancer alleles were ectopically expressed or permanently introduced in the genome of mouse or human cells (2-5). To address the limitation of current models, we have used targeted homologous recombination to introduce (knock-in, KI) a panel of cancer alleles in human somatic cells. Specifically, we focused on EGFR, KRAS, BRAF, and PIK3CA mutated alleles that are found in multiple cancer types. Mutant cells have then been used to study the biochemical and transforming potential of common cancer alleles and to identify genotype-specific ...

Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging has been extensively characterized. We hypothesized that solid tumors could take advantage of proteins such as myoglobin to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species. We therefore sought to establish whether myoglobin might be expressed and functionally regulated in epithelial tumors that are known to face hypoxia and oxidative stress during disease progression. We analyzed the expression of myoglobin in human epithelial cancers at both transcriptional and protein levels; moreover, we investigated the expression levels of myoglobin in cancer cell lines subjected to different conditions, including hypoxia, oxidative stress, and mitogenic stimuli. We provide evidence that human epithelial tumors, including breast, lung, ovary, and colon carcinomas, express high levels of myoglobin from the earliest stages of disease development. In human cancer cells, myoglobin is induced by a variety of signals associated with tumor progression, including mitogenic stimuli, oxidative stress, and hypoxia. This study provides evidence that myoglobin, previously thought to be restricted to myocytes, is expressed at high levels by human carcinoma cells. We suggest that myoglobin expression is part of a cellular program aimed at coping with changed metabolic and environmental conditions associated with neoplastic growth. (Am J

* b b-thalassemias are characterized by an imbalance of globin chains with an excess of a-chains which precipitates in erythroid precursors and red blood cells (RBCs) leading to inefficient erythropoiesis. The severity of the disease correlates with the amount of unpaired achains. Our goal was to develop a simple test for evaluation of the free a-hemoglobin pool present in RBC lysates. Alpha-Hemoglobin Stabilizing Protein (AHSP), the chaperone of a-Hb, was used to trap excess aHb. A recombinant GST-AHSP fusion protein was bound to an affinity micro-column and then incubated with hemolysates of patients. After washing, the a-Hb was quantified by spectrophotometry in the elution fraction. This assay was applied to 54 patients: 28 without apparent Hb disorder, 20 b-thalassemic and 6 a-thalassemic. The average value of free a-Hb pool was 93 ± 21 ppm (ng of free a-Hb per mg of Hb subunits) in patients without Hb disorder, while it varies from 119 to 1,756 ppm, in b-thalassemic patients and correlated with genotype. In contrast, the value of the free a-Hb pool was decreased in a-thalassemic patients (65 ± 26 ppm). This assay may help to characterize b-thalassemia phenotypes and to follow the evolution of the globin chain imbalance (a/b+c ratio) in response to treatment.b-thalassemias (b-thal) are inherited autosomal diseases characterized by a decreased or abolished b-globin chain biosynthesis [1]. During the transition of g to b globin expression in the first year of life, the clinical severity is mainly related to the ratio a/b1g which indicates the level of chain disequilibrium. b-thal are generally classified as minor, intermediate, or major phenotype, and encompass a wide clinical spectrum ranging from asymptomatic carriers to forms that can be lethal in the absence of extensive treatment. Different factors can modulate the severity of this disease [2] such as the magnitude of the decrease of the b globin synthesis, for example hemoglobin E (Hb E b26Glu?Lys) [3], or with the co-inheritance of (i) an a-thalassemia (a-thal) [4], (ii) a triplicated a-globin gene [5] or (iii) a low but variable residual capacity of g globin production which can have the phenotype of a hereditary persistence of fetal Hb (Hb F a 2 g 2 ) [6].The imbalance between a and non a-globin (g1d1b) synthesis was established by radioactive in vitro biosynthesis of globin chains from reticulocytes or of bone marrow of b-thalassemic patients [7][8][9][10]. This imbalance leads to an excess of highly unstable free a-hemoglobin (a-Hb), precipitating on the cell membrane and acting as active oxidants causing apoptosis and inefficient erythropoiesis [1,11]. The severity of b-thal is directly correlated with the degree of imbalance as quantified by the amount of unbound a-globin chain [12]. Many studies have shown that one observes a soluble a chain fraction present in the cytosolic RBC and an insoluble a chain fraction in the membrane of b-thalassemic erythrocytes [13,14]. Because of technical difficulties and the burden of using radioactive materials...