TRANSGENIC PUBLICATIONS
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Abstract:The role of a Harvey-ras oncogene in mammary epithelial neoplasia was examined by
infecting primary cultures of normal mouse mammary epithelial cells with either the Harvey murine
sarcoma virus (psi 2HaMSV) alone or with HaMSV plus a helper virus. The biological effects of
expression of the Ha-ras oncogene were determined by transplanting the infected cells into
gland-cleared mammary fat pads of virgin Balb/c mice. Expression of the Ha-ras oncogene was
correlated with the development of mammary epithelial neoplasms. Cells infected with
replication-defective HaMSV alone formed dysplastic, non-invasive mammary outgrowths. Cells
infected with HaMSV plus a helper virus developed poorly-differentiated, invasive mammary
epithelial tumors. Uninfected cells and cells infected with only the helper virus formed normal
mammary trees. Expression of the mutant viral Ha-ras p21 was detected in dysplastic outgrowths
and tumors but not in normal mammary outgrowths. Use of this transgenic organ system to
genetically alter epithelium of the mouse mammary gland has permitted correlation of expression of
a Ha-ras oncogene with development of mouse mammary neoplasia.
Abstract: Experimental carcinogenesis has led to a concept that defines two discrete stages in the
development of skin tumors: (i) initiation, which is accomplished by using a mutagen that
presumably activates a protooncogene, and (ii) promotion, which is a reversible process brought
about most commonly by repeated application of phorbol esters. We have created a transgenic
mouse strain that carries the activated v-Ha-ras oncogene fused to the promoter of the mouse
embryonic alpha-like, zeta-globin gene. Unexpectedly, these animals developed papillomas at
areas of epidermal abrasion and, because abrasion can also serve as a tumor-promoting event in
mutagen-treated mouse skin, we tested these mice for their ability to respond to phorbol ester
application. Within 6 weeks virtually all treated carrier mice had developed multiple papillomas,
some of which went on to develop squamous cell carcinomas and, more frequently, underlying
sarcomas. We conclude that the oncogene "preinitiates" carrier mice, replacing the
initiation/mutagenesis step and immediately sensitizing them to the action of tumor promoters. In
addition, treatment of the mice with retinoic acid dramatically delays, reduces, and often completely
inhibits the appearance of promoter-induced papillomas. This strain has use in screening tumor
promoters and for assessing antitumor and antiproliferative agents.
Abstract: An immortal mammary epithelial cell line, Comma 1D, and primary cultures of mammary
epithelial cells were used to examine the effects of vHa-ras on mammary tumor development. In
culture, Comma 1D and primary cells were morphologically indistinguishable. Infection with a
replication defective vHa-ras retroviral vector (psi ras) did not alter their in vitro phenotype.
Uninfected Comma 1D cells implanted into gland-cleared mammary fat pads gave rise to dysplastic
outgrowths, while implants of primary cells gave rise to normal gland structures. After psi ras
infection, implants of Comma 1D cells progressed to adenocarcinomas and those of primary cells
resulted in initiated dysplastic outgrowths. High level infection of either cell type with replication
competent HaMSV (psi ras plus helper virus) resulted in in vitro transformation and undifferentiated
in vivo tumors. Thus, in vivo analysis was necessary to detect the observed correlation between
tumorigenic stage and level of infection. In this system, expression of vHa-ras was vital but not
sufficient for mammary tumor initiation and progression, which resulted from an accumulation of
events that did not need to occur in a specific order.
Abstract: The hypothesis that oncogenes influence tumor phenotype was tested by examining
slides from 607 mammary tumors from 407 transgenic mice bearing the ras, myc, and/or neu
oncogenes. Most tumors (91%) had patterns (phenotypes) that could not be classified by Dunn's
standard nomenclature. The nonstandard tumors were described as eosinophilic small cell (SC),
basophilic large cell (LC), or pale intermediate cell (IC). The SC tumor was associated with ras, the
LC was associated with myc, and the IC was associated with neu, with specificities more than .90
and sensitivities ranging from .99 to .48. Thus, the tumor phenotype could be used to predict which
oncogene was present in the animal. The presence of myc in combination with either ras or neu
resulted in the predominance of LC tumors and accelerated tumorigenesis. The combination of ras
and neu resulted in a decreased tumor incidence. Thus, knowledge of the oncogenes that were
present could be used to predict the natural history of the disease.
Abstract: The effect of mammary gland-specific expression of the polyomavirus middle T antigen
was examined by establishing lines of transgenic mice that carry the middle T oncogene under the
transcriptional control of the mouse mammary tumor virus promoter/enhancer. By contrast to most
transgenic strains carrying activated oncogenes, expression of polyomavirus middle T antigen
resulted in the widespread transformation of the mammary epithelium and the rapid production of
multifocal mammary adenocarcinomas. Interestingly, the majority of the tumor-bearing transgenic
mice developed secondary metastatic tumors in the lung. Taken together, these results suggest that
middle T antigen acts as a potent oncogene in the mammary epithelium and that cells that express it
possess an enhanced metastatic potential.
Abstract:
BACKGROUND:
Previous studies have shown associations between overexpression of
int-2 messenger RNA (mRNA) and murine mammary tumors and between amplification of the int-2
genomic locus and human breast cancers. The Int-2 protein (fibroblast growth factor 3) is a member
of the heparin-binding growth factor family of proteins. The export of these growth factors from cells
may depend on the presence of amino terminal sequences containing hydrophobic signal peptides.
Although Int-2 has a putative signal sequence, it is not known whether or how this protein is secreted
from cells.
PURPOSE:
Assuming that the Int-2 protein is secreted from mammary epithelial cells in
a basolateral direction so that it is available to affect adjacent cells, we investigated whether it acts
in a paracrine manner, exerting its effect externally on adjacent cells, or in an autocrine manner,
exerting its effect internally within the same cell.
METHODS: Using in situ hybridization with
35S-labeled RNA antisense probes that specifically detect mRNA coding for the Int-2 protein, we
determined the cell-specific localization of int-2 mRNA expression in the mammary gland of
transgenic FVB/N mice overexpressing int-2 mRNA. Then we transplanted pieces of mammary
epithelial tissue expressing int-2 mRNA into the mammary fat-pad of wild-type, syngeneic animals.
The mammary glands of host animals were examined as whole-mounts and as histologic sections
2-6 months after transplantation. In situ hybridization was used to confirm which cells continued to
express int-2 mRNA following transplantation.
RESULTS:
Int-2 mRNA expression in transgenic
mice was localized to the mammary epithelial cells. Transplants expressing int-2 mRNA were
similar to wild-type transplants in that they had no observable effect on either the growth or the
morphology of host mammary epithelium. Abnormal growth occurred only in transplanted tissue
expressing int-2 mRNA but not in adjacent host mammary epithelium.
CONCLUSION:
Given the
limitations of our experimental system and the limited information available to date on the secretion
of Int-2 protein, these results suggest that, although the Int-2 protein contains a putative signal
peptide, it may act primarily as an autocrine or as an ultra-short-range paracrine growth factor in
mammary epithelium.
Abstract: Overexpression and amplification of the neu (c-erbB2, ERBB2) protooncogene have
been implicated in the development of aggressive human breast cancer. To directly assess the
effect of mammary gland-specific expression of the neu protooncogene, transgenic mice carrying
unactivated neu under the transcriptional control of the mouse mammary tumor virus
promoter/enhancer were established. By contrast to the rapid tumor progression observed in
several transgenic strains carrying the activated neu transgene, expression of unactivated neu in the
mammary epithelium resulted in the development of focal mammary tumors after long latency. The
majority of the mammary tumors analyzed expressed elevated levels of neu-encoded mRNA and
protein. Overexpression of neu in the mammary tumors was also associated with elevated neu
intrinsic tyrosine kinase activity and the de novo tyrosine phosphorylation of several cellular proteins.
Interestingly, many of the tumor-bearing transgenic mice developed secondary metastatic tumors in
the lung. These observations suggest that overexpression of the unactivated neu protein can induce
metastatic disease after long latency.
Abstract: The human T-cell leukemia virus type 1 (HTLV-I) is associated with adult CD4+ T-cell
leukemia (ATL) and tropical spastic paraparesis (TSP). In as much as only a small percentage of
individuals infected with HTLV-I develop either disease, we set out to model a genetic partner for
this virus in an effort to understand and possibly reproduce its pathophysiology. To this end we have
developed a binary set of transgenic mice, one bearing the relatively inactive HTLV-I long terminal
repeat (LTR) positioned to drive the c-myc oncogene and another bearing a fusion transgene
consisting of the immunoglobulin promoter/enhancer driving the gene for the HTLV-I transcription
activator, tax. Alone, the tax construct, though expressed in the thymus, spleen, lung and brain, has
no deleterious effect. Alone, the HTLV-I LTR/c-myc construct is expressed at very low levels in
lymphoid cells and occasionally induces lymphomas in older animals. When these two transgenic
lines are mated, bigenic offspring harboring both transgenes exhibit dramatic tumor formation. As in
the human, these animals develop CD4+ T-cell lymphomas, but they also develop central nervous
system tumors by 25-90 days of age. The syndrome, which is 100% penetrant and lethal, provides
an animal model for adult T-cell lymphoma and a source of cultured cells of neurogenic origin.
Abstract: A transgenic mouse strain with the zeta-globin promoter and the vHa-ras oncogene
develops an array of mesenchymal and epithelial neoplasms described here. The predominate
mesenchymal tumors were dermal spindle cell tumors, which resembled malignant fibrous
histiocytomas found in humans. They were associated with hepatosplenomegaly and developed
beneath squamous papillomas. The hepatosplenomegaly was associated with infiltrates of cells that
tended toward myelocytic or monocytic differentiation. Other epithelial tumors included
keratoacanthomas and squamous cell carcinomas. Squamous cysts, some with squamous cell
carcinomas, of the salivary glands and mammary carcinomas were also found. Odontogenic
tumors, which sometimes differentiated into ameloblastomas, were one of the more unusual tumor
types observed. Other, less frequent tumors were also noted. The tumors described here are a
potentially valuable experimental resource that may lead to an understanding of malignant fibrous
histiocytoma-like lesions, odontogenic tumors, and tumor progression.
Abstract: Transgenic mice expressing the polyomavirus (PyV) middle T oncogene in the mammary
epithelium develop multifocal mammary tumors that metastasize with high frequency. The potent
transforming activity of PyV middle T antigen can, in part, be attributed to its ability to associate with
and to activate a number of c-Src family tyrosine kinases (c-Src, c-Yes, and Fyn). As a first step
toward assessing the role of individual c-Src family tyrosine kinases in PyV middle T
antigen-induced mammary tumorigenesis, we have crossed transgenic mice carrying the mouse
mammary tumor virus (MMTV)/PyV middle T antigen fusion gene with mice bearing a disrupted
c-src proto-oncogene. In contrast to the rapid tumor progression seen in the original MMTV/PyV
middle T antigen strains, mice expressing the transgene in the absence of functional c-Src rarely
developed mammary tumors. After long latency, these mice did eventually develop abnormal
hyperplastic mammary tissue. This growth disturbance was correlated with elevated expression of
the PyV middle T antigen and the activation of the PyV middle T antigen-associated c-Yes tyrosine
kinase. However, transgenic mice expressing the PyV middle T antigen in the mammary epithelium
of wild-type or Yes-deficient mice developed multifocal mammary tumors with comparable kinetics.
Taken together, these findings suggest that c-Src tyrosine kinase activity is required for PyV middle
T antigen-induced mammary tumorigenesis and also illustrate an in vivo genetic approach to the
dissection of mitogenic signal transduction pathways.
Abstract: Leukemia inhibitory factor (LIF) is a cytokine involved in embryonic and hematopoietic
development. To investigate the effects of LIF on the lymphoid system, we generated a line of
transgenic mice that expresses diffusible LIF protein specifically in T cells. These mice display two
categories of phenotype that were not previously attributed to LIF overexpression. First, they display
B cell hyperplasia, polyclonal hypergammaglobulinemia and mesangial proliferative
glomerulonephritis, defects similar to those described for transgenic mice overexpressing the
functionally related cytokine, interleukin-6. Secondly, the LIF transgenic mice display novel thymic
and lymph node abnormalities. In the thymus, cortical CD4+CD8+ lymphocytes are lost, while
numerous B cell follicles develop. Peripheral lymph nodes contain a vastly expanded CD4+CD8+
lymphocyte population. Furthermore, the thymic epithelium is profoundly disorganized, suggesting
that disruption of stroma-lymphocyte interactions is responsible for many observed defects.
Transplantation of transgenic bone marrow into wild type recipients transfers both the thymic and
lymph node defects. However, transplantation of wild type marrow into transgenic recipients rescues
the lymph node abnormality, but not the thymic defect, indicating the thymic epithelium is irreversibly
altered. Our observations are consistent with a role for LIF in maintaining a functional thymic
epithelium that will support proper T cell maturation.
Abstract: Physical associations between cyclins, viral oncogenes and tumour suppressor genes
imply a central role for cyclins in growth control. Cyclin D1 was identified as a candidate oncogene
(PRAD1) in tumour-specific DNA rearrangements and is suspected to be a contributor to several
types of neoplasms including breast cancer. Cyclin D1 also rescues G1 cyclin-defective
Saccharomyces cerevisiae, and is a growth-regulated gene. Despite evidence suggesting that
cyclin D1 is an oncogene, its ability to transform cells directly in culture remains controversial. To
evaluate its potential to deregulate growth in vivo in a physiologically relevant tissue we
overexpressed cyclin D1 in mammary cells in transgenic mice. We report here that overexpression
of cyclin D1 resulted in abnormal mammary cell proliferation including the development of mammary
adenocarcinomas. We conclude that overexpression of cyclin D1 deregulates cell proliferation and
can induce tumorigenic changes in mammary tissues, suggesting that cyclin D1 indeed plays an
important oncogenic role in breast cancer.
Abstract: Mammary hyperplasias, dysplasias and tumors have been described in many strains of
transgenic mice. Many of the transgenes produce characteristic disturbances of growth,
development and neoplasia. The disturbances can now be classified into groups. A classification of
transgenic tumors and an atlas illustrating some characteristic examples of common morphological
changes in transgenic mammary tissues is provided.
Abstract: Activation of the c-Src tyrosine kinase has been implicated as an important step in the
induction of mammary tumors in both mice and humans. To directly assess the effect of mammary
gland-specific expression of activated c-Src, we established transgenic mice that carry a
constitutively activated form of c-src under transcriptional control of the murine mammary tumor virus
long terminal repeat. Female mice derived from several independent transgenic lines lactate poorly
as a consequence of an impairment in normal mammary epithelial development. In addition to this
lactation defect, female mice frequently develop mammary epithelial hyperplasias, which
occasionally progress to frank neoplasias. Taken together, these observations suggest that
expression of activated c-Src in the mammary epithelium of transgenic mice is not sufficient for
induction of mammary tumors.
Abstract: Expression of the activated neu oncogene in transgenic mice has been associated with
both the synchronous (single-step) and the stochastic (multistep) transformation of the mammary
epithelium. To determine the basis for these conflicting observations, additional strains of
transgenic mice carrying the activated neu oncogene under the transcriptional control of the mouse
mammary tumor virus promoter/enhancer were produced. Activated neu transgene expression, as
measured by in situ hybridization and ribonuclease protection assays, resulted in rapid conversion
of the normal mammary epithelium to malignant phenotype in three independent strains of mice.
Expression of the transgene in male mice led to epithelial hyperplasia of the epididymis and male
infertility but not malignancy. These results indicate that tissue context is an important parameter in
malignant progression and that expression of appropriate levels of activated neu is sufficient for
rapid production of mammary tumors in transgenic mice.
Abstract:
Receptor tyrosine kinases are important in cell signal transduction and proliferation.
Abnormal expression of tyrosine kinases often leads to malignant transformation. C-met is a
tyrosine kinase receptor and its ligand is hepatocyte growth factor (HGF). HGF/c-met plays diverse
role in regulation of cell growth, shape and movement. Constitutively activated met, such as tpr-met,
is a potent oncogene in vitro, but its carcinogenic role in vivo remains unclear. Our study
demonstrates that expression of tpr-met leads to development of mammary tumors and other
malignancies in transgenic mice, and suggests that deregulated met expression may be involved in
mammary carcinogenesis.
Abstract
Platelets, essential for vascular integrity and hemostasis, fragment from polyploid megakaryocytes, characterized by their endomitotic cell cycle. We studied the influence of overexpression of c-myc oncogene on megakaryopoiesis and endomitosis in vivo, using transgenic mice carrying c-myc fused to the estrogen receptor under the control of the platelet factor 4 (PF4) megakaryocyte-specific promoter. The rationale behind this strategy was to obtain controlled overexpression of an active c-Myc, depending on the estrogen level in the mouse circulation. Analysis of these transgenic mice revealed that the bone marrow of female transgenic mice of estrogen-injected male transgenic mice, but not of age-matched transgenic males nor nontransgenic females, contained frequent immature myeloid cells and an increased number of megakaryocytes. Deregulated expression of c-Myc shifted the normal ploidy profile of megakaryocytes due to a significant increase in proliferating megakaryocytes and a decrease in the fraction of ploidizing cells. These transgenic mice represent a novel in vivo model for a Myc-induced myeloproliferative disorder which can be controlled.
Abstract: Signal transduction regulation was interrupted in four independent transgenic mice by expressing the polyomavirus
middle T (PyMT) gene, to affect the growth and development of the prostate gland and other urogenital tissues.
Expression of PyMT was directed to these tissues by a novel, androgen-inducible expression vector based on the
rat C3(1) gene. Epithelial growth disturbances ranging from tissue hyperplasia, cellular dysplasia to invasive
carcinoma were observed in the ventral and dorsal prostate. Similar abnormalities were seen in the coagulating
gland, epididymis, and vas deferens. The abnormalities were recognized by histological changes within the ventral
and dorsal prostates were recognized by histologic disorganization, nuclear pleomorphism, increased mitosis and
abnormal ploidy. Transgene transcription was detected in all affected tissues, demonstrating that the C3(1)-based
vector targeted androgen sensitive urogenital tissues, especially the prostate. Therefore, the expression of a
protein altering signal transduction pathways disrupts urogenital growth and development..
23. Tulchin, N., F.S. Lee, L. Ornstein, J. Strauchen and R.D. Cardiff. Immunohistologic c-
myc protein in benign breast disease and cancer. Int. J. Oncology 9:419-425, 1996.
(ABSTRACT)(ILLUSTRATIONS)
Abstract
The MTV/myc transgenic mouse model has permitted the analysis of the effects of exogenously introduced myc genes, fused to the murine mammary tumor virus promoter, on the development of mammary tumors. We have used a sensitive immunohistochemical method for studying the Myc protein in eleven mammary tumors from two independent MTV/myc transgenic lines. The tumors found in these animals included: four large cell adenocarcinomas (LC), two type A tumors of Dunn, and one mixed type A and C tumor of Dunn, and four unclassified adenocarcinomas. The highest levels of staining for Myc were found in the epithelial cell nuclei of mammary tumors; the nuclei in the transgenic mesenchyme did not contain detectable antigen. Morphologically normal transgenic epithelium had foci of stained nuclei. No detectable nuclear Myc stain was found in non-transgenic mammary glands of FVB mice. This work supports a role of the Myc protein in mammary tumorigenesis and suggests a correlation between high levels of Myc and histopathology.
24. Muller, W.J., C..L. Arteaga, S.K. Muthuswamy, P.M. Siegel, M.A. Webster, R.D. Cardiff, K.S. Meise, F. Li, S.A. Halter and R. Coffey. Synergistic interaction of the Neu protooncogene and TGFa in the mammary epithelium of transgenic mice. Molecular and Cellular Biology. 16:5726-5736, 1996.
(ABSTRACT)(ILLUSTRATIONS)
Abstract
Transgenic mice expressing either the neu proto-oncogene or transforming growth factor (TGF-a) in the mammary epithelium develop spontaneous focal mammary tumors that occur after a long latency. Since the epidermal growth factor receptor (EGFR) and Neu are capable of forming heterodimers that are responsive to EGFR ligands such as TGF-a, we examined whether coexpression of TGF-a and Neu in mammary epithelium could cooperate to accelerate the onset of mammary tumors. To test this hypothesis, we interbred separate transgenic strains harboring either a mouse mammary tumor virus/TGF-a or a mouse mammary tumor virus/neu transgene to generate bitransgenic mice that coexpress TGF-a and neu in the mammary epithelium. Female mice coexpressing TGF-a and neu developed multifocal mammary tumors which arose after a significantly shorter latency period than either parental strain alone. The development of these mammary tumors was correlated with the tyrosine phosphorylation of Neu and the recruitment of c-Src to the Neu complex. Immunoprecipitation and immunoblot analysis with EGFR- and Neu-specific antisera, however, failed to detect physical complexes of these two receptors. Taken together, these observations suggest that Neu and TGF-a cooperate in mammary tumorigenesis through a mechanism involving Neu and EGFR transactivation.
Last Updated: November 27, 1996
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