Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-01-30
2003-02-18
Yucel, Remy (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S010000, C800S025000
Reexamination Certificate
active
06521815
ABSTRACT:
BACKGROUND OF THE INVENTION
A majority of human cancers originate from epithelial tissue. A common cancer of epithelial origin is nonmelanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), with more than 700,000 new cases diagnosed each year in the United States. Similar cancers are also seen in non-human animals such as domesticated animals and pets, including cats and dogs. BCC is rarely life-threatening because it is slow growing and is mostly localized. Unlike BCC, SCC metastasizes at a rate of 2% to 6% over several years after initial diagnosis. A highly malignant form invades and destroys tissue, and then metastasizes, initially to a regional lymph node before more distant organs such as the lung or brain are affected. SCC is commonly encountered in a number of epithelial tissues, including the oral cavity, esophagus, larynx, bronchi, intestines, colon, genital tract, and skin. Early detection using reliable biomarkers is desired, as are rationally designed drugs for effectively preventing and treating aggressive, metastatic SCC.
As such, there is a need for a good animal model system for studying how metastatic squamous cell carcinoma develops, progresses and can be treated. To date, no such model exists. Classically, tumor cells are injected into the tail vein of either immunocompromised or syngeneic mice. While this assay can suitably model the later metastatic stages, it does not model the early genesis, invasion and angiogenic stages of malignant progression, especially as it relates to complex interactions between tumor and host, especially at the tissue site where the carcinoma originated. Moreover, the role of the immune system in metastatic progression cannot be analyzed when immunocompromised mice are used.
Murine skin model systems are still essential contributors to our understanding of the multi-step nature of chemically-induced carcinogenesis. In the multistage mouse skin carcinogenesis model, biochemical events unique to initiation, promotion, or progression can be studied and related to cancer formation. In that model, the NMSC that is most often induced is squamous cell carcinoma. Although squamous cell carcinoma of mouse skin invades the dermal region, the incidence of malignant metastatic conversion is rare and requires a long latency period of approximately a year.
Several protocols are used to develop mouse skin tumors in laboratory animals. In a common initiation-promotion protocol, mouse skin is treated with an initiating agent (7,12-dimethylbenz[a]anthracene; DMBA) and then with a potent tumor promoter (12-O-tetradecanoylphorbol-13-acetate; TPA). In this protocol, mice develop mostly benign papillomas, more than 90% of which regress after TPA treatment is stopped. Only a small percentage of papillomas progress to invasive, but non-metastatic, SCC. The initiation-promotion protocol has been further modified to enhance the conversion of skin papillomas to carcinomas, yet metastatic potential is not increased.
A major intracellular receptor for TPA is the ubiquitous enzyme protein kinase C (PKC), an important signal transduction pathway component for controlling cell proliferation and tumorigenesis. It has been suggested that PKC activation may play a role in promoting mouse skin tumor formation. However, several groups have demonstrated that repeated applications of TPA depress PKC activity and protein levels. These results indicate that both loss of PKC activity and degradation of PKC could be important for mouse skin tumor promotion by TPA.
On the basis of the structural similarities and cofactor requirements, the eleven known PKC isoforms are grouped into three subfamilies: (1) the conventional PKCs (&agr;, &bgr;I, &bgr;II, and &ggr;), which depend upon Ca
2+
, phosphatidylserine (PS), and diacylglycerol (DAG) or TPA; (2) the nPKCs (&egr;, &dgr;, &eegr;, and &thgr;), which require only PS and DAG/TPA; and (3) the atypical PKCs (&igr;/&lgr; and &xgr;), which retain PS dependence but have no requirement for Ca
2+
or DAG/TPA for activation. PKC&mgr;, which is usually classified as a nPKC, is not easily grouped with any of the other isoforms.
The roles of PKC&agr; and PKC
&dgr;
isoforms in the mouse skin tumor initiation/promotion protocol were assessed in FVB/N transgenic mice expressing an T7-epitope-tagged PKC&agr; (T7-PKC&agr;) or PKC&dgr; (T7-PKC&dgr;) under the control of the human keratinocyte-specific K14 promoter/enhancer. Transgenic expression of T7-PKC&agr; did not affect tumor promotion susceptibility. Transgenic expression of T7-PKC&dgr; in the epidermis (~8-fold increase) suppressed the formation of both skin papillomas and carcinomas by 70%.
PKC&egr; may play an important role in cellular growth regulation. TPA binds to and activates PKC&egr;. Activated PKC&egr; may be important for the survival of small cell lung carcinoma cell lines in which the catalytic fragment of PKC&egr; is constitutively expressed. Overexpression of PKC&egr; in Rat-6 or NIH-3T3 fibroblasts increases growth rate, anchorage independence, and tumor formation in nude mice. PKC&egr; overexpression also transforms non-tumorigenic rat colonic epithelial cells and suppresses apoptosis of interleukin-3 dependent human myeloid cells induced by removal of interleukin-3.
The role of PKC&egr; in mouse skin tumor promotion and epidermal cell growth and differentiation remains unclear. Treatment of the mouse skin with TPA leads to a general reduction in PKC activity that persists for at least 4 days. Acute TPA treatment decreases PKC&bgr; and &eegr; protein levels, but has little or no effect on the levels of PKC&agr;, &dgr;, or &egr;. PKC&agr;, &bgr;, and &dgr; activity levels were reduced after acute or repeated TPA treatments, but PKC&egr; activity was not examined. DMBA/TPA-induced papillomas exhibit decreased cytosolic levels of PKC&agr; and &bgr;II protein, but insignificant alterations in the levels of PKC&dgr;, &egr;, or &xgr; protein. When cultured mouse skin keratinocytes are induced to differentiate by increasing Ca
2+
, PKC&egr;, &dgr;, and a translocate to the membrane fraction, suggesting a role for activation of these isoforms in keratinocyte differentiation.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that an FVB/N mouse that expresses more PKC&egr; in its epidermis than in the epidermis of a wild-type FVB/N mouse is a useful model for development and treatment of skin cancer, particularly squamous cell carcinoma, in human and non-human mammalian animals. In a preferred embodiment, the mouse expresses at least about 5-fold more epidermal PKC&egr; than wild-type FVB/N mice, with more preferred embodiments having still higher levels of epidermal PKC&egr;.
The present invention is also summarized in that an FVB/N mouse that expresses PKC&egr; in its epidermis at a level higher than a wild-type FVB/N mouse, where the level is sufficiently high to induce metastatic growth, is a useful model for development and treatment of metastatic skin cancer, particularly for metastatic squamous cell carcinoma, in humans. Notably the mice constitute a model system for developing and treating highly malignant metastatic squamous cell carcinoma. A level sufficiently high is more than 5-fold higher than in wild-type FVB/N mice, and is preferably at least about 12-fold higher, and still more preferably at least about 15-fold higher, and most preferably at least about 18-fold higher.
The present invention is also summarized in that a method for inducing squamous cell carcinomas in the aforementioned mice includes the steps of treating the mouse with a skin tumor initiating chemical agent, then treating the mouse repeatedly with an skin tumor promotion chemical agent for a time sufficient to induce squamous cell carcinomas and then screening the treated mice to identify those mice in which squamous cell carcinoma is induced. In a related embodiment, the skin tumor initiating agent can be DMBA and the skin tumor promotion agent can be TPA. After treatment according to the method, the mic
Jansen Aaron P.
Reddig Peter J.
Verma Ajit K.
Pappu Sita
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
Yucel Remy
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