Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts.

BACKGROUND: Rapid, robust and reversible induction of transgene expression would significantly facilitate cancer gene therapy as well as allow the in vivo functional study of newly discovered genes in tumor formation and progression. The popularity of the ecdysone inducible gene switch system has led us to investigate whether such a system can successfully regulate gene expression in a syngeneic tumor system in vivo. RESULTS: MBT-2 and Panc02 carcinoma cells were transfected with components of a modification of the ecdysone switch system driving firefly luciferase (F-Luc). In vitro luciferase expression +/- ecdysone analog GS-E indicated a robust induction with minimal baseline activity and complete decay after 24 hours without drug. In vitro selection of MBT-2 transfected cell clones which had complete absence of F-Luc expression in the absence of stimulation but which expressed this gene at high levels in response to GS-E were chosen for in vivo evaluation. Tumors from engineered MBT-2 cells were grown to 5 mm in diameter prior to GS-E administration, animals euthanized and tumors removed at 6, 12 and 24 hours after GS-E administration and assayed for F-Luc activity. GS-E resulted in a maximal induction of F-Luc activity at 6 hours in tumor tissue with almost complete reversion to control levels by 12 hours. CONCLUSIONS: This study is the first demonstration that robust and reversible transgene expression in tumors is feasible using the ecdysone system, allowing future rapid in vivo functional characterization of gene function or gene therapy applications.

Analysis of the FHIT gene and its product in squamous cell carcinomas of the head and neck.

The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine if FHIT alterations play a role in human squamous cell carcinogenesis of the head and neck (HNSCC), we examined the gene and its product by RT-PCR, SSCP, Northern, Southern, and Western blot analysis in primary HNSCC and/or HNSCC cell lines. Three of 32 tumor samples lacked detectable expression of FHIT by RT-PCR but showed amplification of a control gene of similar size. One of 29 primary tumors and 2/9 HNSCC cell lines exhibited aberrant transcripts generated by RT-PCR methods using one set of 40 cycles of amplification. FHIT mRNA expression was absent in seven HNSCC cell lines but detectable in primary keratinocytes by Northern analysis. Using specific polyclonal antiserum to the full-length FHIT protein in immunoblot analyses, 4/9 cell lines analysed showed no expression of pFhit, two exhibited low levels of expression, and three expressed a putative truncated pFhit. One of 15 tumors analysed also exhibited an overexpressed truncated protein. PCR/SSCP and Southern analysis of one cell line DNA that expressed a truncated protein indicated that it sustained homozygous loss of FHIT exon 5. Our results suggest that alterations in FHIT at the DNA, RNA, and protein levels exist at a low but significant frequency in HNSCCs. Further studies regarding the potential biological activity of FHIT are needed to clarify the role of this gene in HNSCC tumorigenesis.

Similar incidence of K-ras mutations in lung carcinomas of FVB/N mice and FVB/N mice carrying a mutant p53 transgene.

Mutated p53 genes are capable of complementing activated ras genes in the transformation of primary rat embryo fibroblasts in vitro. Mutations in both genes have also been found in several human cancers, including lung carcinomas. We generated transgenic mice containing a p53 construct with a missense mutation in exon 5 (ala135val) to study the role of p53 mutations in lung tumorigenesis, and to facilitate identification of other genetic events that might complement p53 mutations in mouse lung carcinogenesis. The p53 transgenic lines exhibited a higher frequency of lethal lung tumors than the parental FVB/N strain. We examined the spontaneously-arising lung carcinomas from mice expressing the mutated p53 transgene for K-ras mutations using single-stranded conformation polymorphism (SSCP) and/or direct sequencing approaches. Fifteen of 29 (52%) carcinomas contained mutations in the K-ras oncogene. Six of 15 of the K-ras mutations were in codon 61 and 9/15 were in codon 12. Subsequent analysis of spontaneous lung carcinomas from mice of the FVB/N parental strain showed that 9/12 (75%) carcinomas examined contained K-ras mutations. Two of these were in codon 12, one in codon 13, and 6 were in codon 61. These results demonstrate that the frequency of ras mutations does not differ between the p53 FVB/N transgenic mice and their parental FVB/N strain but suggest that a high frequency of mutations K-ras can be correlated with lung tumorigenesis in both groups of mice.

Chronic ethanol ingestion modifies liver microsomal phosphatidylserine inducing resistance to hydrolysis by exogenous phospholipase A2.

Chronic ethanol ingestion leads to the acquisition of a tolerance to membrane lipid disordering, a lowered partition coefficient to hydrophobic compounds and a resistance to the hydrolysis of the phospholipids by exogenous phospholipase A2. Anionic phospholipids have been implicated as being responsible for the resistance to lipid disordering and a number of modifications to these phospholipids are known to occur as a result of chronic ethanol-ingestion. In this study the basis of the resistance to phospholipase A2 in hepatic microsomes was investigated. It was found that chronic ethanol-induced modifications to each of the major phospholipid classes was responsible to some extent for the resistance to phospholipase A2, however, PS was particularly potent considering it is a compositionally minor constituent. The effect was interpreted as a reduced ability to activate the phospholipase A2 since PS acts as an essential activator of phospholipase A2 (along with PI). Fatty acid analysis revealed that the chronic ethanol-treatment resulted in a elevated level of docosahexaenoate with a parallel reduction in arachidonate in phosphatidylserine. Lipid packing and organization is important in the regulating the level of exogenous phospholipase A2 activity but the activity was not found to correlate with lipid order of different phosphatidylserine species. It is concluded that subtle differences in the molecular species arrangement or disposition around the enzyme may be responsible for the altered phospholipase A2 interaction with the membrane induced by chronic ethanol-treatment. One implication of this study is that other anionic phospholipid dependent membrane proteins, of which there are many known examples, may also be modified as a result of chronic ethanol-ingestion.

Effect of increasing the level of omega-3 fatty acids on rat skeletal muscle sarcoplasmic reticulum.

The effect of dietary supplementation with fish oil as compared to corn oil on the lipid dynamics and calcium ATPase activity of rat skeletal sarcoplasmic reticulum was examined. After four-week supplementation with fish oil, the levels of eicosapentaenoic (20:5 omega 3), docosapentaenoic (22:5 omega 3) and docosahexaenoic (22:6 omega 3) acids in the total lipids were 5.3, 5.5 and 28.1% of the total fatty acids, respectively. In contrast, with corn oil only 22:6 was found (8.9%). The level of these fatty acids in phosphatidylethanolamine from the membranes of animals fed fish oil was 4.2 (20:5), 5.4 (22:5) and 49.1% (22:6); and for phosphatidylcholine it was 5.4 (20:5), 4.6 (22:5) and 17.4% (22:6). Again, in corn oil fed animals, only 22:6 was found in appreciable amounts, namely 28.3% in phosphatidylethanolamine and 1.8% in phosphatidylcholine. The steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess lipid order and was found to be only slightly less for membranes from animals supplemented with fish oil (0.120) as compared to those supplemented with corn oil (0.124). The calcium ATPase was found to be unaffected by supplementation consistent with the observed modest changes in lipid order as well as with suggestions that the enzyme is relatively insensitive to the level of unsaturation. It could be argued that if large increases in fatty acyl polyunsaturation in mammalian cell membranes would lead to marked alterations in bulk membrane lipid motional properties, this may not be in the interest of preserving physiological function.(ABSTRACT TRUNCATED AT 250 WORDS)