Non-invasive detection of a small number of bioluminescent cancer cells in vivo.

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.

Persistent induction of cytochrome P450 (CYP)1A enzymes by 3-methylcholanthrene in vivo in mice is mediated by sustained transcriptional activation of the corresponding promoters.

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens. Cytochrome P450 (CYP)1A enzymes play key roles in the metabolic activation of PAHs to carcinogenic metabolites. We previously showed persistent induction of CYP1A enzymes by 3-methylcholanthrene (MC) in vivo in rodents. In this study, we tested the hypothesis that MC elicits persistent induction of CYP1A1 and 1A2 in vivo by mechanisms entailing sustained transcriptional activation of the corresponding promoters. Adult male wild type (WT) (Cd-1) mice, transgenic mice expressing the human CYP1A1 promoter or the mouse CYP1A2 promoter were treated with the vehicle corn oil (CO) or the carcinogenic PAH, 3-methylcholanthrene (MC), once daily for 4days, and luciferase reporter gene expression was determined at 1, 8, 15, and 22days after MC withdrawal by bioluminescent imaging. Pulmonary and hepatic endogenous expression of CYP1A1 and 1A2 was also determined at the enzymatic, protein, and mRNA levels. The major findings were that MC elicited marked enhancement in the luciferase expression in the CYP1A1-luc as well CYP1A2-luc transgenic mice that was sustained for up to 22days, the magnitude of induction being more pronounced in the CYP1A1-luc mice. MC also caused persistent induction of endogenous CYP1A1 and 1A2 expression in the WT, CYP1A1-luc, and 1A2-luc mice for up to 22days. In conclusion, our results support the hypothesis that MC elicits sustained CYP1A1 and 1A2 expression by sustained transcriptional activation of the corresponding promoters. Thus, these novel transgenic models should be very useful for further understanding of the molecular mechanisms of persistent CYP1A induction, in relation to PAH-mediated carcinogenesis.

Noninvasive bioluminescence imaging of normal and spontaneously transformed prostate tissue in mice.

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

A Cyp1a2-luciferase transgenic CD-1 mouse model: responses to aryl hydrocarbons similar to the humanized AhR mice.

Here we describe a transgenic mouse model [Crl:CD-1(ICR)BR-Tg(Cyp1a2-luc)Xen] using luciferase as a reporter for Cyp1a2 gene regulation. An 8.4-kilobase mouse Cyp1a2 promoter driving the firefly luciferase gene was microinjected into single-cell-stage CD-1 mouse embryos. A transgenic mouse line was selected based on basal and induced levels of the transgene in mouse liver by an in vivo bioluminescent imaging method. The basal levels of the luciferase reporter in liver were expressed much higher than other tissues, which correlated well with the endogenous Cyp1a2 mRNA tissue distribution. Male signals were about 23-fold higher than females in liver. However, the Cyp1a2 mRNA showed no gender difference. When mice were challenged with xenobiotics, the liver luciferase signal was induced to various degrees. At the doses we used, the relative effects were phenobarbital > 2,3,7,8-tetrachlorodibenzo-p-dioxin > 3-methylcholanthrene > benzo[a]pyrene and beta-naphthoflavone. Induction of the Cyp1a2-luc reporter was generally consistent with the endogenous Cyp1a2 mRNA. However, phenobarbital induction was unexpectedly higher, while beta-naphthoflavone induction of the reporter was much lower than that of the endogenous Cyp1a2 gene. Induction of the Cyp1a2-luc transgene by aryl hydrocarbons (Ah) in the CD-1 background was much less than that found in the Ah responsive C57BL/6 mice, while being similar to the nonresponsive DBA/2 strain. Sequence analysis of the CD-1 Ah receptor (AhR) cDNA clones demonstrated that consensus sequence was identical to some of the Ah-responsive strains such as BALB/C and CBA/J mice. The 104-kD AhR protein was not detectable in CD-1 mice, while the 97-kD AhR was detected in the C57BL/6 mice by Western blot using an AhR antibody. Low expression of the AhR in CD-1 mice could be in part responsible for low responsiveness to Ah compounds. The findings demonstrated the outbred CD-1 mouse is a low-responsive strain, and the Cyp1a2-luc transgenic CD-1 mice can be used for studying the regulation of the mouse Cyp1a2 gene in an Ah low-responsive strain in real time using the bioluminescent imaging approach.

Differential regulation of the human CYP3A4 promoter in transgenic mice and rats.

Previously we described a transgenic mouse model [FVB/NTg(CYP3A4-luc)Xen] using a reporter construct consisting of 13 kilobases of the human CYP3A4 promoter driving the firefly luciferase gene in the inbred FVB/N mouse strain. Here we report regulation of the same CYP3A4-luc reporter gene in a transgenic outbred mouse strain (CD-1) and in a transgenic rat (Sprague-Dawley). Basal reporter expression and responses to several xenobiotics in the transgenic CD-1 mice [CD-1/Crl-Tg(CYP3A4-luc)Xen] were similar to those in the transgenic FVB/N mice. In both mouse backgrounds, the basal levels of the reporter were higher in male compared with female, and in the FVB/N strain there was greater induction for all drugs in male compared with female; however, in the CD-1 background this gender difference for induction was not obvious. In contrast with transgenic mice, transgenic rats [SD/Tac-Tg(CYP3A4-luc)Xen] expressed the luciferase reporter at higher basal levels in female compared with male rats. Responses to some compounds were much greater in rats than in mice, and the kinetics of induction was different with peak induction occurring later in the rat compared with the mouse. Our results suggest that the human CYP3A4 promoter is regulated differently in transgenic mice and rats in some aspects.

A transgenic mouse model with a luciferase reporter for studying in vivo transcriptional regulation of the human CYP3A4 gene.

Cytochrome p450 3A4 (CYP3A4) plays an important role in drug metabolism, and the enzymatic activity of CYP3A4 contributes to many adverse drug-drug interactions. Here we describe a transgenic mouse model that is useful in monitoring the in vivo transcriptional regulation of the human CYP3A4 gene. A reporter construct consisting of 13 kilobases of the human CYP3A4 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line [FVB/N-Tg(CYP3A4-luc)Xen]. Reporter gene expression was assessed using an in vivo imaging system (IVIS) in anesthetized mice. Basal expression of the reporter was highest in liver and kidney, and moderate in the duodenum in male transgenic mice, whereas the basal luciferase activity was highest in the duodenum and lower in kidney and liver in females. Injections of pregnenolone, phenobarbital, rifampicin, nifedipine, dexamethasone, 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), and clotrimazole resulted in a time-dependent induction of luciferase expression, primarily in liver, that peaked at 6 h post injection. The greatest induction was found with clotrimazole, dexamethasone, and PCN, whereas the lowest induction followed pregnenolone, phenobarbital, and rifampicin injection. In general, male mice responded to these drugs more strongly than did females. Our results suggest that the human CYP3A4 promoter functions in transgenic mice and that this in vivo model can be used to study transcriptional regulation of the CYP3A4 gene.