Estrogen receptor-positive mammary tumorigenesis in TGFalpha transgenic mice progresses with progesterone receptor loss.

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFalpha) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFalpha mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/-) p53 mice did not acquire mammary lesions, p53+/- mice carrying the NRL-TGFalpha transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFalpha expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR-, and when combined with a heterozygous p53 genotype, ER-/PR-.

Dysregulation of mammary Stats 1,3 and 5 and PRL receptors by overexpression of TGFalpha.

Mammary TGFalpha overexpression results in delayed involution and eventually mammary cancer in transgenic mice. We hypothesized that STATs and PRL receptors (PRLR), critical regulators of mammary function, are altered in these animals and may contribute to this phenotype. We examined these factors late in the first pregnancy (d.18) and during normal involution (d.4 post-lactation) in WAP-TGFalpha transgenic mice and non-transgenic controls. Long form PRLR mRNA in WAP-TGFalpha glands at both pregnant d.18 and d.4 post-lactation was significantly reduced compared to controls, and PRLR-S3 failed to rise during involution. Total and pTyr STAT 1,3,5a and 5b also were altered. STAT 3 was higher at both times in WAP-TGFalpha glands. STAT 5a and 5b were lower at late pregnancy, but higher post-lactation; however, pTyr(694) STAT 5 was abnormally low at both times. Thus overexpression of TGFalpha has direct or indirect effects on both STATs and PRL responsiveness in vivo, which may reflect mechanisms of TGFalpha-induced mammary epithelial abnormalities.

A strategy to identify differentially expressed genes using representational difference analysis and cDNA arrays.

Representational difference analysis (RDA) combined with cDNA arrays is an effective approach to identify differentially expressed genes. To identify differentially expressed genes in c-Myc transgenic mouse liver, we compared the virtues of probing commercially available cDNA arrays with either radiolabeled cDNA pools or radiolabeled difference products (DP2) derived from RDA using c-Myc transgenic and normal mouse liver. Probing commercial and custom arrays with DP2 products led to the identification of transcripts of low abundance that were missed when the arrays were initially probed with PCR-amplified cDNA pools. Although DP2 probes also detected abundant transcripts that are highly differentially expressed, they failed to identify abundant transcripts with low differential expression that were detected with cDNA pools. The combined use of radiolabeled cDNA and DP2 products to probe arrays allows a more comprehensive identification of differentially expressed transcripts that are abundant or rare. Our method has the additional benefit of eliminating false-positive transcripts that lack true differential expression and frequently contaminate DP2 pools. Using this method we identified 16 differentially expressed genes in c-Myc transgenic liver, one of which is novel.

Hepatocyte transplantation into diseased mouse liver. Kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes.

To examine the process of liver repopulation by transplanted hepatocytes, we developed transgenic mice carrying a mouse major urinary protein-urokinase-type plasminogen activator fusion transgene. Expression of this transgene induced diffuse hepatocellular damage beginning at 3 weeks of age, and homozygous mice supported up to 97% parenchymal repopulation by healthy donor hepatocytes transplanted into the spleen. Using this transplantation model, we determined that 1) a mean of 21% of splenically injected hepatocytes engraft in liver parenchyma; 2) a mean of 6.6% of splenically injected hepatocytes (or one-third of engrafted cells) can give rise to proliferating hepatocyte foci; 3) transplanted cells in proliferating foci display an initial cell-doubling time of 28 hours, and focus growth continues through a mean of 12 cell doublings; 4) hepatocytes isolated from young and aged adult mice display similar focus repopulation kinetics; 5) the extent of repopulated parenchyma remains stable throughout the life of the recipient mouse; and 6) tetraploid and octaploid hepatocytes can support clonal proliferation.

Timing of hepatocyte entry into DNA synthesis after partial hepatectomy is cell autonomous.

After surgical removal of two-thirds of the liver, remaining hepatocytes replicate and restore hepatic mass within 2 weeks. This process must be initiated by signals extrinsic to the hepatocyte, but it remains unclear whether subsequent events leading to DNA synthesis (S phase) are regulated by circulating or locally produced growth factors (a noncell autonomous response), or by a program intrinsic to the hepatocyte itself (a cell autonomous response). To identify the type of mechanism regulating passage to S, we exploited the difference between rat and mouse hepatocytes in the timing of DNA synthesis after partial hepatectomy, which peaks 12-16 h earlier posthepatectomy in rat compared with mouse. Four groups of animals received two-thirds partial hepatectomies: rats, mice, mice with chimeric livers composed of both transplanted rat hepatocytes and endogenous mouse hepatocytes, and mice with chimeric livers composed of both transplanted and endogenous mouse hepatocytes. Following two-thirds partial hepatectomy, both donor and endogenous hepatocytes in mouse/mouse chimeric livers displayed kinetics of DNA synthesis characteristic of the mouse, indicating that transplantation per se did not affect the response to subsequent partial hepatectomy. In contrast, rat hepatocytes in chimeric mouse livers displayed rat kinetics despite their presence in a mouse host. Thus, factors intrinsic to the hepatocyte must regulate the timing of entry into DNA synthesis. This result defines the process as cell autonomous and suggests that locally or distantly produced cytokines or growth factors may have a permissive but not an instructive role in progression to S.

Highly invasive transitional cell carcinoma of the bladder in a simian virus 40 T-antigen transgenic mouse model.

Transitional cell carcinoma (TCC), a neoplasm of urinary bladder urothelial cells, generally appears in either of two forms, papillary non-invasive or invasive TCC, although intermediate forms can occur. Each has a distinctive morphology and clinical course. Altered expression of the p53 and pRb genes has been associated with the more serious invasive TCC, suggesting that the loss of activity of these tumor suppressor proteins may have a causal role in this disease. To test this hypothesis directly, transgenic mice were developed that expressed the simian virus 40 large T antigen (TAg) in urothelial cells under the control of the cytokeratin 19 gene (CK19) regulatory elements. In one CK19-TAg lineage, all transgenic mice developed highly invasive bladder neoplasms that resembled invasive human bladder TCCs. Stages of disease progression included development of carcinoma in situ, stromal invasion, muscle invasion, rapid growth, and, in 20% of affected mice, intravascular lung metastasis. Papillary lesions never were observed. Western blot analysis indicated that TAg was bound to both p53 and pRb, which has been shown to cause inactivation of these proteins. Our findings support suggestions that (i) inactivation of p53 and/or pRb constitutes a causal step in the etiology of invasive TCC, (ii) papillary and invasive TCC may have different molecular causes, and (iii) carcinoma in situ can represent an early stage in the progression to invasive TCC.

Cellular origin of regenerating parenchyma in a mouse model of severe hepatic injury.

Several treatments in rodents, including administration of the alkylating agent dipin, followed by two-thirds partial hepatectomy in mice combine destruction of liver parenchyma with hepatocyte mitoinhibition. These treatments induce proliferation of bile epithelial-like cells (termed oval cells), development of foci composed of small hepatocytes, and eventual replacement of damaged parenchyma by healthy hepatocytes. It has been proposed that these oval cells represent transitional cells in a nonhepatocytic liver facultative stem cell lineage that can give rise to the small hepatocyte foci, and that these foci eventually become confluent and replace liver parenchyma. In this study, we used in vivo cell lineage marking in genetically chimeric livers to test the hypothesis that hepatocytes can serve as the precursor cell type to the small hepatocyte foci that develop in mouse liver after treatment with dipin plus partial hepatectomy. Although we do not exclude the possibility that some small hepatocyte foci may be stem cell-derived, we demonstrate that hepatocyte-derived foci are present after dipin-induced liver damage in mice.

Seeing is believing: non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography.

The ability to monitor reporter gene expression in living animals and in patients will permit longitudinal examinations both of somatically transferred DNA in experimental animals and patients and of transgenic constructs expressed in experimental animals. If investigators can non-invasively monitor the organ and tissue specificity, the magnitude and the duration of gene expression from somatically transferred DNA and from transgenes, conceptually new experimental paradigms will be possible. If clinicians can non-invasively monitor the location, extent and duration of somatically transferred genes, they will be better able to determine the correlations between expression of therapeutic genes and clinical outcomes. We have developed two reporter gene systems for in vivo reporter gene imaging in which the protein products of the reporter genes sequester positron-emitting reporter probes. The "PET reporter gene" dependent sequestration of the "PET reporter probes" is subsequently measured in living animals by Positron Emission Tomography (PET). We describe here the principles of PET reporter gene/PET reporter probe in vivo imaging, the development of two imaging systems, and the validation of their ability to non-invasively, quantitatively and repetitively image reporter gene expression in murine viral gene transfer and transgenic models.

Hepatocyte transplantation in a model of toxin-induced liver disease: variable therapeutic effect during replacement of damaged parenchyma by donor cells.

To provide long-term therapy in patients with severe toxin-induced hepatic parenchymal damage, donor hepatocytes would need to replicate and replace a large portion of the damaged parenchyma. Using a mouse model developed to reproduce this type of hepatic injury, we found that hepatocyte transplantation only slightly improved survival after transplantation despite the fact that many non-survivors showed moderate liver repopulation by donor cells. Perhaps accounting for this outcome, donor parenchyma in non-survivors did not have typical lobular organization. These results indicate that the re-creation of functional parenchyma by transplanted hepatocytes requires time, during which donor cells proliferate and then establish normal parenchymal architecture.

Transforming growth factor alpha- and c-myc-induced mammary carcinogenesis in transgenic mice.

The growth factor transforming growth factor alpha (TGFalpha) and the nuclear transcription factor c-myc often are overexpressed by human breast cancer cells. To produce models of breast disease with these etiologies, mice were generated that carried TGF-alpha- or c-myc-encoding transgenes. Transgene targeting employed the whey acidic protein (WAP) gene promoter, which is expressed in pregnant and lactating mammary epithelial cells. Non-virgin WAP-TGFalpha transgenic mice displayed accelerated mammary development during pregnancy, delayed post-parturient mammary involution, a progressive increase in the number of hyperplastic alveolar nodules (HANs), and development of mammary carcinoma with a mean latency of 9 months. Non-virgin WAP-c-myc transgenic mice displayed accelerated mammary gland development during pregnancy and development of mammary carcinomas with a latency of 8 months. Bitransgenic mice carrying both WAP-TGFalpha and WAP-c-myc displayed a dramatic acceleration of tumor development. These models identify the overexpression of TGFalpha or c-myc as etiological factors in the development of mammary neoplasia and demonstrate the increased severity of disease when both molecular alterations are present in the same cell.

Cryopreserved mouse hepatocytes retain regenerative capacity in vivo.

BACKGROUND & AIMS: Substitution of hepatocyte transplantation for whole liver transplants in selected individuals with liver disease could significantly expand the number of patients to benefit from use of scarce donor livers. However, successful hepatocyte transplantation may require that donor cells retain normal functional and proliferative capabilities and that they be readily available. Banking of cryopreserved hepatocytes would fulfill the latter requirement. Cryopreservation protocols have been developed that minimize hepatocyte injury and allow preservation of metabolic activity. The aim of this study was to assess cryopreserved hepatocyte proliferative capacity in vivo after thawing. METHODS: Fresh and frozen/thawed mouse hepatocytes were transferred separately into the livers of recipient mice with transgene-induced liver disease, an environment that is permissive for clonal expansion of donor cell populations. Fresh and cryopreserved donor cells were compared for their ability to proliferate and replace damaged parenchyma. RESULTS: Although cryopreservation decreased hepatocyte viability, individual viable frozen/thawed hepatocytes demonstrated clonal replicative potential identical to that of fresh hepatocytes. Even after storage for 32 months in liquid nitrogen, transplanted hepatocytes constituting 0.1% of total adult hepatocyte number could repopulate a mean of 32% of recipient liver parenchyma. CONCLUSIONS: These findings suggest that cryopreserved hepatocytes represent an appropriate source of cells for therapeutic hepatocyte transplantation.

Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor alpha.

BACKGROUND & AIMS: The progenitor cells responsible for transforming growth factor (TGF)-alpha-induced pancreatic ductal metaplasia and neoplasia remain uncharacterized. During pancreatic development, differentiated cell types arise from ductal progenitor cells expressing the Pdx1 homeodomain transcription factor. The aims of this study were, first, to evaluate the role of Pdx1-expressing stem cells in MT-TGFalpha transgenic mice, and second, to further characterize cell proliferation and differentiation in this model. METHODS: To assess Pdx1 gene expression in normal and metaplastic epithelium, we performed in vivo reporter gene analysis using heterozygous Pdx1(lacZ/+) and bigenic Pdx1(lacZ/+)/MT-TGFalpha mice. RESULTS: Pdx1(lacZ/+)/MT-TGFalpha bigenics showed up-regulated Pdx1 expression in premalignant metaplastic ductal epithelium. In addition to Pdx1 gene activation, TGF-alpha-induced metaplastic epithelium demonstrated a pluripotent differentiation capacity, as evidenced by focal expression of Pax6 and initiation of islet cell neogenesis. The majority of Pdx1-positive epithelial cells showed no expression of insulin, similar to the pattern observed during embryonic development. CONCLUSIONS: Overexpression of TGF-alpha induces expansion of a Pdx1-expressing epithelium characterized by focal expression of Pax6 and initiation of islet neogenesis. These findings suggest that premalignant events induced by TGF-alpha in mouse pancreas may recapitulate a developmental program active during embryogenesis.

Ubiquitous expression of marker transgenes in mice and rats.

The ability to unambiguously mark a cell's genotype is essential for studies in which genetically distinct cell populations must be distinguished from one another in vivo. One approach to this challenge has been the creation of transgenic mice expressing a transgene marker that is easily detectable, with no background staining. Multiple transgenic mouse strains bearing constructs with different combinations of promoter elements and coding sequences have been described, each with its own advantages and limitations. In this report we describe the use of an 800-bp promoter fragment isolated from the beta(geo) integration site in ROSA26 mice to target expression of two marker genes. We demonstrate that the ROSA26 promoter directs ubiquitous expression of human placental alkaline phosphatase and enhanced green fluorescent protein during embryonic and postnatal development in mouse and rat. We further demonstrate the general utility of these transgenes for marking donor cells in transplantation studies.

Liver disease and compensatory growth: unexpected lessons from genetically altered mice.

Over the last decade, several animal models have been established that permit exploration of liver biology and disease. Although these models have been developed using diverse strategies, including transgene targeting, homozygous gene disruption and administration of hepatotoxic chemicals, each approach creates an animal with hepatocyte damage, resulting in an hepatic microenvironment that supports proliferation of healthy hepatocytes. These models have been used to demonstrate: (1) the remarkable ability of adult hepatocytes to clonally proliferate in response to liver growth signals, (2) the effectiveness of transplanted donor hepatocytes in repopulating damaged liver parenchyma, and (3) the feasibility of reconstituting liver with xenogeneic hepatocytes. This paper reviews the development and use of these models, and outlines their potential future application to the study of hepatic stem cells, therapy of liver disease and hepatic toxicology.

Reduced susceptibility of mice overexpressing transforming growth factor alpha to dextran sodium sulphate induced colitis.

BACKGROUND: Transforming growth factor alpha (TGF-alpha) knockout mice have increased susceptibility to dextran sodium sulphate (DSS) induced colitis. AIM: To substantiate the findings that TGF-alpha is a key mediator of colonic mucosal protection and/or repair mechanisms by evaluating the susceptibility of mice overexpressing TGF-alpha to DSS induced colitis. METHODS: TGF-alpha overexpression was induced in transgenic mice by ZnSO4 administration in drinking water (TG+). Three groups were used as controls: one transgenic group without ZnSO4 administration (TG-), and two non-transgenic littermate groups receiving ZnSO4 (Non-TG+) or only water (Non-TG-). Acute colitis was induced in all groups by administration of DSS (5%, w/v) in drinking water for six days and libitum. RESULTS: About 35-39% of the entire colonic mucosa was destroyed in Non-TG-, Non-TG+, and TG- animals compared with 9% in TG+ mice. the crypt damage score was 18.7 (0.9), 18.2 (1.0), 18.9 (0.8), and 6.8 (1.5) (means (SEM)) in Non-TG-, Non-TG+, TG-, and TG+ mice respectively. Mucin and bromodeoxyuridine staining were markedly enhanced in colons of TG+ mice compared with controls, indicating increased mucosal protection and regeneration. CONCLUSIONS: The significantly reduced susceptibility of mice overexpressing TGF-alpha to DSS further substantiates that endogenous TGF-alpha is a pivotal mediator of protection and/or healing mechanisms in the colon.

Altered differentiation of hepatocytes in a transgenic mouse model of hepatocarcinogenesis.

Transgenic mice carrying the SV40 T antigen (TAg) gene, which develop hepatocellular and biliary cell tumors by 4 mo of age, show ductular structures in the neonatal liver. Coexpression of c-myc with TAg increases the extent and persistence of ductular lesions and also accelerates tumor development. To analyze possible links between altered gene expression and cell differentiation and to determine the relationship between the ductular structures and tumor development in these mice, ductular cells in single (TAg) and bitransgenic (TAg x c-myc) mice were characterized for biliary and hepatocellular differentiation, transgene expression, and proliferation activity. The results show that the ductular cells in these transgenic mice have characteristics of biliary cells, including basement membrane formation, positive laminin staining, and bile duct-specific lectin (Dolichos biflorus agglutinin and peanut agglutinin) binding, and characteristics of hepatocytes, including albumin expression and ultrastructural features such as round nuclei with 1 or 2 nucleoli and well-developed cytoplasmic organelles. However, differences in transgene expression and cell proliferation between the ductular cells and nonductular hepatocytes were not apparent. Thus, the ductular cells could not be defined as tumor progenitor cells in these mouse livers. However, this model suggests that manipulation of gene expression can alter differentiation of hepatic parenchymal cells.

Interactive effects of c-myc and transforming growth factor alpha transgenes on liver tumor development in simian virus 40 T antigen transgenic mice.

To analyze the effects of c-myc and transforming growth factor alpha (TGFalpha) on hepatocarcinogenesis induced by simian virus 40 T antigen (TAg), livers from single and bitransgenic mice, 3 to 11 mice per line, were examined morphologically 1 to 8 weeks after birth. Mice carrying c-myc or TGFalpha alone exhibited centrilobular hypertrophy and increased apoptosis (c-myc mice only) of hepatocytes after 3 or 4 weeks of age, but no detectable changes in cell proliferation or proliferative lesions were observed in either line during the 8 weeks. Mice carrying TAg alone exhibited increased cell proliferation, apoptosis, and dysplasia of hepatocytes with notably high mitotic and apoptotic indices as major changes before development of putative preneoplastic lesions after 4 weeks of age and neoplastic lesions after 6 weeks. In bitransgenic mice coexpressing c-myc or TGFalpha with TAg, nonproliferative lesions and mitotic and apoptotic indices were similar to those in mice carrying TAg alone. In TAg x c-myc bitransgenic mice, however, both preneoplastic and neoplastic lesions developed sooner and grew more rapidly than those in TAg mice, whereas in TAg x TGFalpha bitransgenic mice, rapid tumor growth was the principle observation. Because of the effects of transgene coexpression, livers from TAg x c-myc and TAg x TGFalpha mice had multiple tumors as early as 3 and 6 weeks of age, respectively. The results indicate cooperative functions of c-myc and TGFalpha with TAg during development and/or growth of liver tumors in vivo.

A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes.

Synthesis of mouse metallothionein (MT)-I and MT-II is transcriptionally induced by the synthetic glucocorticoid, dexamethasone (DEX) or both in vivo as well as in numerous cell lines. However, the location(s) of a glucocorticoid response element (GRE) has not been described. The observation that a marked MT-I gene, as well as heterologous genes, when placed in the context of 17 kb of flanking sequence from the MT locus, are inducible by DEX and lipopolysaccharide in transgenic mice renewed the search for the GRE. Analysis of a series of deletion constructs from this 17-kb region in cultured cells identified a single 455-bp region that conferred DEX induction on a reporter gene. This 455-bp region contains two GREs that bind to the glucocorticoid receptor as assessed by gel mobility shift. Deletion of this fragment from the 17-kb flanking region eliminates the DEX responsiveness of reporter genes. The two GREs, which are located approximately 1 kb upstream of the MT-II gene and approximately 7 kb upstream of the MT-I gene, are necessary for induction of both genes and can function independently of elements within the proximal promoter region of either gene.

Albumin gene expression during mouse odontogenesis.

Albumin protein is present in developing teeth of several species. Oligomer primers and cRNA probes specific for albumin were designed to perform RT-PCR, and for in situ hybridization, respectively. In situ hybridization failed to reveal albumin expression in any tooth cells, however, albumin PCR products were amplified from tissues adhering to the roots of developing teeth from four-week-old mice. It is concluded that this source is not the primary source of albumin protein found in developing enamel, because of the location and level of expression of albumin mRNA in periodontal tissue.

Inhibition of mammary gland involution is associated with transforming growth factor alpha but not c-myc-induced tumorigenesis in transgenic mice.

Deregulated expression of transforming growth factor alpha (TGF-alpha) or c-myc has been implicated in the genesis of human breast cancer. To better characterize the role of these molecules in this disease, we generated transgenic mice that express TGF-alpha or c-myc under control of the mouse whey acidic protein (WAP) promoter. We then compared the resulting mammary gland neoplasia in these mice and in previously described mice expressing a metallothionein-driven TGF-alpha transgene. Nonvirgin female mice in all transgenic lineages developed mammary tumors with 100% incidence but variable latency. Among TGF-alpha lines, mean survival time correlated with the level of transgene expression, and the average life spans of high-expressing WAP-TGF-alpha and WAP-c-myc mice were similarly reduced. The majority of TGF-alpha-induced tumors were relatively well-differentiated adenomas and adenocarcinomas; in contrast, WAP-c-myc tumors were poorly differentiated, solid carcinomas with a minority of adenocarcinomas. Most TGF-alpha and all c-myc-induced tumors were transplantable, but lung metastases were infrequently observed in all transgenic lines. WAP-TGF-alpha-induced tumors, in marked contrast to those induced by WAP-c-myc, displayed frequent induction of cyclin D1 mRNA, suggesting that expression of this gene may complement that of TGF-alpha during mammary tumor development. Expression of TGF-alpha also induced precocious development of pregnant glands and delayed or inhibited mammary involution. As a result, multiparious MT-TGF-alpha and especially WAP-TGF-alpha females accumulated large numbers of hyperplastic alveolar nodules that resembled the more differentiated TGF-alpha-induced tumors. Finally, coexpression of WAP-c-myc and WAP-TGF-alpha transgenes markedly decreased tumor latency, increased tumor growth, and even induced mammary tumors in virgin female and male mice. These findings provide further evidence for the importance of deregulated TGF-alpha expression in multistage carcinogenesis, and they suggest that in the mammary gland the mechanism of TGF-alpha-induced transformation may depend on postlactational survival of differentiated epithelium. They also provide evidence of a potent tumorigenic collaboration between TGF-alpha and c-myc in mammary epithelium.