Tracing the geographic origins of weedy Ipomoea purpurea in the southeastern United States.

Ipomoea purpurea (common morning glory) is an annual vine native to Mexico that is well known for its large, showy flowers. Humans have spread morning glories worldwide, owing to the horticultural appeal of morning glory flowers. Ipomoea purpurea is an opportunistic colonizer of disturbed habitats including roadside and agricultural settings, and it is now regarded as a noxious weed in the Southeastern US. Naturalized populations in the Southeastern United States are highly polymorphic for a number of flower color morphs, unlike native Mexican populations that are typically monomorphic for the purple color morph. Although I. purpurea was introduced into the United States from Mexico, little is known about the specific geographic origins of US populations relative to the Mexican source. We use resequencing data from 11 loci and 30 I. purpurea accessions collected from the native range of the species in Central and Southern Mexico and 8 accessions from the Southeastern United States to infer likely geographic origins in Mexico. Based on genetic assignment analysis, haplotype composition, and the degree of shared polymorphism, I. purpurea samples from the Southeastern United States are genetically most similar to samples from the Valley of Mexico and Veracruz State. This supports earlier speculation that I. purpurea in the Southeastern United States was likely to have been introduced by European colonists from sources in Central Mexico.

Nucleotide sequence diversity of floral pigment genes in Mexican populations of Ipomoea purpurea (morning glory) accord with a neutral model of evolution.

The common morning glory (Ipomoea purpurea) is an annual vine native to Central and Southern Mexico. The genetics of flower color polymorphisms and interactions with the biotic environment have been extensively studied in I. purpurea and in its sister species I. nil. In this study, we examine nucleotide sequence polymorphism in 11 loci, 9 of which are known to participate in a pathway that produces floral pigments. A sample of 30 I. purpurea accessions from the native range of Central and Southern Mexico comprise the data, along with one accession from each of the two sister species I. alba and I. nil. We observe moderate levels of nucleotide sequence polymorphism of ~1%. The ratio of recombination to mutation parameter estimates (ρ/θ) of ~2.5 appears consistent with a mixed-mating system. Ipomoea resequencing data from these genic regions are noteworthy in providing a good fit to the standard neutral model of molecular evolution. The derived silent site frequency spectrum is very close to that predicted by coalescent simulations of a drift-mutation process, and Tajima's D values are not significantly different from expectations under neutrality.

Targeting KRAS mutation-bearing lung cancer in vivo by pulmonary surfactant-adenovirus-mediated gene transfer.

Pulmonary surfactant has been used as a carrier to deliver a therapeutic virus to dysfunctional lung cells that reside within an intricate lung structure. To investigate whether pulmonary surfactant enhances the efficacy of intratracheal instillation of a therapeutic virus to target KRAS mutation-bearing lung cancer in vivo, we developed a recombinant adenovirus that induces cell death only in lung cancer cells and injected the adenovirus into a mouse model of KRAS mutation-positive lung cancer intratracheally with and without surfactant. A therapeutic adenovirus that induces cell death only in lung cancer cells was constructed by combining a cancer-specific human telomerase reverse transcriptase (hTERT) promoter fused to CCAAT/enhancer-binding protein alpha (CEBPα) with a modified lung-specific Clara cell-specific 10-kDa protein (CC10) promoter fused to cytotoxic adenovirus type 5 early region 1A (E1A). CEBPα is induced only in cancer cells and activates the CC10 promoter, which in turn induces cytotoxic E1A, and causes cell death only in lung cancer cells in vitro. This adenovirus was intratracheally administered to the model mice (CCSP-rtTA/Tet-op-K-Ras4bG12D bitransgenic mice) in the presence and absence of pulmonary surfactant. Intratracheally administered therapeutic adenovirus with pulmonary surfactant spread to airways, as well as to the alveolar region of the lung, and caused a reduction of lung tumors developed. The therapeutic adenovirus without pulmonary surfactant spread only to airways and was ten-fold less effective in tumor reduction. Here, we demonstrate that pulmonary surfactant is an efficient tool to intratracheally deliver a therapeutic virus to treat KRAS mutation-positive lung cancer in vivo.

Inhibition of Myc effectively targets KRAS mutation-positive lung cancer expressing high levels of Myc.

Myc is an oncogenic transcription factor that promotes tumorigenesis. Recently, a dominant negative form of Myc (Omomyc) was shown to cause regression of lung tumors in a mouse model of lung cancer caused by KRAS mutation, suggesting that Myc might be a potential therapeutic target to treat the KRAS lung cancer. However, it is not yet known whether Omomyc can also inhibit the growth of human lung tumors that carry a similar KRAS mutation. In the present study, we demonstrate that Omomyc induces cell death of KRAS-mutated human lung adenocarcinoma A549 cells in vitro and in vivo. However, Omomyc does not induce cell death in human lung adenocarcinoma H441 cells that also carry the KRAS mutation. Interestingly, A549 cells express high levels of Myc, while H441 cells do not. Co-expression of exogenous Myc with Omomyc in H441 cells induces cell death, indicating that Omomyc requires high levels of Myc to induce cell death in KRAS mutation-positive lung adenocarcinoma. Here, we show for the first time that KRAS mutation-positive lung cancer displaying high levels of Myc could be treated by inhibiting Myc transactivation function.

Adenovirus-mediated cancer gene therapy and virotherapy (Review).

Gene therapy and virotherapy are among the approaches currently used to treat malignant tumors. Gene therapy and virotherapy use a specific therapeutic gene that causes death in cancer cells. In early attempts at gene therapy, therapeutic genes were driven by ubiquitous promoters such as the CMV promoter, which induce non-specific toxicity to normal cells and tissues in addition to the cancer cells. Recently, novel cancer- and/or tissue-specific promoter systems have been developed to target cancer cells but not normal cells including stem cells. In this review, we describe cancer and/or tissue-specific gene therapy systems for the treatment of cancer. In particular, we will discuss three systems for gene therapy and virotherapy: i) tissue-specific promoter systems, ii) cancer-specific promoter systems, and iii) oncolytic virotherapy. We will also discuss the major challenges of cancer-targeting vector systems and future directions in this area.

Drug-regulatable cancer cell death induced by BID under control of the tissue-specific, lung cancer-targeted TTS promoter system.

Gene therapy and virotherapy are among the approaches currently being used to treat lung cancer. The success of cancer gene therapy depends on treatments where different types of tumors can be selectively targeted and destroyed without affecting normal cells and tissue. Previously, we described a promoter system (TTS) that we designed that is specifically targeted to lung cancer cells but which does not affect other types of cells including stem cells. In our study, we have enhanced the utility of the TTS system by inserting the pro-apoptotic gene BH3 domain interacting death agonist (Bid) into the TTS promoter system (TTS/Bid) to create a drug regulatable lung cancer-specific gene therapy. A recombinant adenoviral vector was used to introduce TTS/Bid (Ad-TTS/Bid) into lung cancer cells. BID expression and apoptosis occurred in A549 pulmonary adenocarcinoma cells but little Bid expression or apoptosis occurred in MCF7 breast cancer cells or in normal human lung fibroblasts. The use of cisplatin enhanced the processing of full length BID to t-BID which significantly increased lung cancer-specific cell death. In in vivo experiments, intraperitonal injection of cisplatin enhanced the antitumor effects of the vector in a lung cancer xeno-graft mouse model. Moreover, dexamethasone effectively suppressed exogenous BID expression and the antitumor effect of Ad-TTS/Bid both in vitro and in vivo. Here, we describe the efficacy of the use of cisplatin and dexamethasone with the anti lung cancer promoter system (Ad-TTS/Bid) for a safe and effective gene therapy against advanced lung cancer.

Malignant pleural mesothelioma-targeted CREBBP/EP300 inhibitory protein 1 promoter system for gene therapy and virotherapy.

Gene therapy and virotherapy are one of the approaches used to treat malignant pleural mesothelioma. To improve the efficiency of targeting malignant mesothelioma cells, we designed a novel system using the promoter of the CREBBP/EP300 inhibitory protein 1 (CRI1), a gene specifically expressed in malignant pleural mesothelioma. Four tandem repeats of the CRI1 promoter (CRI1(-138 4x)) caused significantly high promoter activity in malignant pleural mesothelioma cells but little promoter activity in normal mesothelial cells and normal fibroblasts. The recombinant adenoviral vector expressing proapoptotic BH3-interacting death agonist or early region 1A driven by the CRI1(-138 4x) promoter induced cell death in malignant mesothelioma cells but not in normal cells. Moreover, these viruses showed antitumor effects in a mesothelioma xenograft mouse model. Here, we describe a novel strategy to target malignant mesothelioma using the CRI1(-138 4x) promoter system.

Pulmonary adenocarcinoma-targeted gene therapy by a cancer- and tissue-specific promoter system.

Gene therapy is one of the approaches used to treat lung cancer. The benefit of cancer gene therapy is that different types of tumors can be selectively targeted by tumor-specific expression of therapeutic genes that include an apoptosis gene to destroy the tumor. Previously, we described a promoter (TTS promoter) that we designed that is specifically targeted to lung cancer cells but not to other types of cancer or normal cells including stem cells. In this pursuit, we further characterize the specificity of the TTS promoter in four types of lung cancer cells (squamous cell lung carcinoma, pulmonary adenocarcinoma, small-cell lung carcinoma, large-cell lung carcinoma). The TTS promoter is highly active only in pulmonary adenocarcinoma cells but not in the other three types of lung cancer cells. The specificity seems to be derived from transcription factor thyroid transcription factor 1-associating cofactors that affect human surfactant protein A1 promoter activity in pulmonary adenocarcinoma. We inserted the proapoptotic gene Bcl-2-associated X protein (Bax) into the TTS promoter (TTS/Bax). The TTS/Bax selectively causes BAX expression and cell death in pulmonary adenocarcinoma but not in other cells. Cell death caused by the BAX expression was also observed in pulmonary adenocarcinoma that is resistant to the anticancer drug gefitinib (epidermal growth factor receptor tyrosine kinase inhibitor). BAX expression and cell death can be suppressed by dexamethasone (a glucocorticoid) treatment through negative glucocorticoid elements in the TTS promoter. Here we report a drug-controllable TTS/Bax system targeting pulmonary adenocarcinoma.

Tumour suppressor p53 down-regulates the expression of the human hepatocyte nuclear factor 4alpha (HNF4alpha) gene.

The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumour suppressor protein p53. We have previously shown that p53 inhibits the transactivation function of HNF (hepatocyte nuclear factor) 4alpha1, a nuclear receptor known to be critical for early development and liver differentiation. In the present study we demonstrate that p53 also down-regulates expression of the human HNF4alpha gene via the proximal P1 promoter. Overexpression of wild-type p53 down-regulated endogenous levels of both HNF4alpha protein and mRNA in Hep3B cells. This decrease was also observed when HepG2 cells were exposed to UV irradiation or doxorubicin, both of which increased endogenous p53 protein levels. Ectopically expressed p53, but not a mutant p53 defective in DNA binding (R249S), down-regulated HNF4alpha P1 promoter activity. Chromatin immunoprecipitation also showed that endogenous p53 bound the HNF4alpha P1 promoter in vivo after doxorubicin treatment. The mechanism by which p53 down-regulates the P1 promoter appears to be multifaceted. The down-regulation was partially recovered by inhibition of HDAC activity and appears to involve the positive regulator HNF6alpha. p53 bound HNF6alpha in vivo and in vitro and prevented HNF6alpha from binding DNA in vitro. p53 also repressed stimulation of the P1 promoter by HNF6alpha in vivo. However, since the R249S p53 mutant also bound HNF6alpha, binding HNF6alpha is apparently not sufficient for the repression. Implications of the p53-mediated repression of HNF4alpha expression in response to cellular stress are discussed.

Development of a novel beta-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures.

Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med. 343 (2000) 230-238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407-415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259-266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A, 97 (2000) 7999-8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691-1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. In order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel beta-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3x. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3x construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and beta-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3x shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel beta-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting beta-like insulin-producing cells that have differentiated from stem cells or other progenitor cells.

Genes that determine flower color: the role of regulatory changes in the evolution of phenotypic adaptations.

A central goal of evolutionary genetics is to trace the causal pathway between mutations at particular genes and adaptation at the phenotypic level. The proximate objective is to identify adaptations through the analysis of molecular sequence data from specific candidate genes or their regulatory elements. In this paper, we consider the molecular evolution of floral color in the morning glory genus (Ipomoea) as a model for relating molecular and phenotypic evolution. To begin, flower color variation usually conforms to simple Mendelian transmission, thus facilitating genetic and molecular analyses. Population genetic studies of flower color polymorphisms in the common morning glory (Ipomoea purpurea) have shown that some morphs are subject to complex patterns of selection. Striking differences in floral color and morphology are also associated with speciation in the genus Ipomoea. The molecular bases for these adaptive shifts can be dissected because the biosynthetic pathways that determine floral pigmentation are well understood and many of the genes of flavonoid biosynthesis have been isolated and extensively studied. We present a comparative analysis of the level of gene expression in Ipomoea for several key genes in flavonoid biosynthesis. Specifically we ask: how frequently are adaptive shifts in flower color phenotypes associated with changes in regulation of gene expression versus mutations in structural genes? The results of this study show that most species differences in this crucial phenotype are associated with changes in the regulation of gene expression.

Tracing floral adaptations from ecology to molecules.

Flowers have long fascinated humans. The scientific study of floral biology unifies many diverse areas of research, ranging from systematics to ecology, and from genetics to molecular biology. Despite this unity, few plant species offer the experimental versatility to encompass all levels of biological investigation in a single system. An exception is the morning glory genus Ipomoea, in which a broad picture of floral evolution, ranging from ecology to molecular biology, is emerging.