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1.
Oncogene ; 36(18): 2529-2542, 2017 05 04.
Article in English | MEDLINE | ID: mdl-27991921

ABSTRACT

To study ROCK2 activation in carcinogenesis, mice expressing 4-hydroxytamoxifen (4HT)-activated ROCK2 (K14.ROCKer) were crossed with mice expressing epidermal-activated rasHa (HK1.ras1205). At 8 weeks, 4HT-treated K14.ROCKer/HK1.ras1205 cohorts exhibited papillomas similar to HK1.ras1205 controls; however, K14.ROCKer/HK1.ras1205 histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma (wdSCC), exhibiting p53 loss, increased proliferation and novel NF-κB expression. By 12 weeks, K14.ROCKer/HK1.ras1205 wdSCCs exhibited increased NF-κB and novel tenascin C, indicative of elevated rigidity; yet despite continued ROCK2 activities/p-Mypt1 inactivation, progression to SCC required loss of compensatory p21 expression. K14.ROCKer/HK1.ras1205 papillomatogenesis also required a wound promotion stimulus, confirmed by breeding K14.ROCKer into promotion-insensitive HK1.ras1276 mice, suggesting a permissive K14.ROCKer/HK1.ras1205 papilloma context (wound-promoted/NF-κB+/p53-/p21+) preceded K14.ROCKer-mediated (p-Mypt1/tenascin C/rigidity) malignant conversion. Malignancy depended on ROCKer/p-Mypt1 expression, as cessation of 4HT treatment induced disorganized tissue architecture and p21-associated differentiation in wdSCCs; yet tenascin C retention in connective tissue extracellular matrix suggests the rigidity laid down for conversion persists. Novel papilloma outgrowths appeared expressing intense, basal layer p21 that confined endogenous ROCK2/p-Mypt1/NF-κB to supra-basal layers, and was paralleled by restored basal layer p53. In later SCCs, 4HT cessation became irrelevant as endogenous ROCK2 expression increased, driving progression via p21 loss, elevated NF-κB expression and tenascin C-associated rigidity, with p-Mypt1 inactivation/actinomyosin-mediated contractility to facilitate invasion. However, p21-associated inhibition of early-stage malignant progression and the intense expression in papilloma outgrowths, identifies a novel, significant antagonism between p21 and rasHa/ROCK2/NF-κB signalling in skin carcinogenesis. Collectively, these data show that ROCK2 activation induces malignancy in rasHa-initiated/promoted papillomas in the context of p53 loss and novel NF-κB expression, whereas increased tissue rigidity and cell motility/contractility help mediate tumour progression.


Subject(s)
Carcinoma, Squamous Cell/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Skin Neoplasms/genetics , Tumor Suppressor Protein p53/genetics , p21-Activated Kinases/genetics , rho-Associated Kinases/genetics , Animals , Carcinogenesis/genetics , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/pathology , Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Crosses, Genetic , Gene Expression Regulation, Neoplastic , Humans , Keratinocytes/pathology , Keratinocytes/virology , Mice , NF-kappa B/genetics , Papilloma/genetics , Papilloma/pathology , Skin Neoplasms/pathology , Tamoxifen/administration & dosage , Tamoxifen/analogs & derivatives
2.
Oncogene ; 33(32): 4132-43, 2014 Aug 07.
Article in English | MEDLINE | ID: mdl-24037529

ABSTRACT

To investigate tumour progression mechanism in transgenic mouse skin carcinogenesis, inducible PTEN ablation (Δ5PTEN(flx)) was targeted to the epidermis of mice expressing activated ras(Ha)/fos oncogenes (HK1.ras and HK1.fos). RU486-treated HK1.ras/fos-Δ5PTEN(flx) epidermis exhibited significant keratinocyte proliferation resulting in hyperplasia and proliferating cysts. While HK1.ras/fos-Δ5PTEN(flx) papillomatogenesis was accelerated, malignant conversion was delayed and tumours exhibited well-differentiated squamous cell carcinoma (wdSCC) histotypes, suggesting inhibition of early-stage malignant progression. Immediate elevated p53/p21 expression was observed in HK1.ras/fos-Δ5PTEN(flx) hyperplasia, cysts and papillomas, and while malignant conversion required p53 loss, elevated p21 expression persisted in most wdSCCs to limit further progression, unless p21 was also lost and wdSCC progressed to more aggressive carcinomas. In contrast, TPA-promoted (that is, c-fos-activated) bi-genic HK1.ras-Δ5PTEN(flx) cohorts lost p53/p21 expression during early papillomatogenesis and rapidly produced poorly differentiated carcinomas (pdSCCs) with high BrdU-labelling and elevated cyclin D1/E2 expression levels, indicative of a progression mechanism driven by failures in cell-cycle control. Intriguingly, HK1.ras/fos-Δ5PTEN(flx) wdSCCs did not exhibit similar failures, as western and immunofluorescence analysis found downregulated cyclin E2 whenever p21 persisted; further, while westerns detected elevated cyclin D1, immunofluorescence identified reduced expression in proliferative basal layer nuclei and a redistributed expression profile throughout p21-positive wdSCC keratinocytes. These data demonstrate that rapid early epidermal responses to ras(Ha)/fos/ΔPTEN co-operation involve induction of p53/p21 to alter differentiation and divert excessive proliferation into cyst formation. Further, despite three potent oncogenic insults p53 loss was required for malignant conversion, and following p53 loss persistent, p53-independent p21 expression possessed the potency to limit early-stage malignant progression via cyclin D1/E2 inhibition.


Subject(s)
Cyclin D1/antagonists & inhibitors , Cyclins/antagonists & inhibitors , PTEN Phosphohydrolase/physiology , Proto-Oncogene Proteins c-fos/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Skin Neoplasms/metabolism , Tumor Suppressor Protein p53/physiology , Animals , Cell Cycle , Cell Differentiation , Cell Proliferation , Gene Expression Regulation, Neoplastic , Keratinocytes/cytology , Mice , Mice, Transgenic , PTEN Phosphohydrolase/genetics , Signal Transduction , Skin Neoplasms/genetics , Tetradecanoylphorbol Acetate/chemistry
3.
Phys Med Biol ; 47(23): 4271-85, 2002 Dec 07.
Article in English | MEDLINE | ID: mdl-12502049

ABSTRACT

Photon migration in a randomly inhomogeneous, highly scattering and absorbing semi-infinite medium with a plane boundary is considered by a Monte Carlo (MC) technique. The employed MC technique combines the statistical weight scheme and real photon paths simulation, allowing the exclusion of the energy conservation problem. The internal reflection of the scattered radiation on the medium interface is taken into account by allowing the trajectories of photon packets to be split into reflected and transmitted parts. The spatial photon sensitivity profile (SPSP), spatially resolved diffuse reflectance and angular and spatial photon detector weight distributions are considered in terms of Fresnel's reflection/refraction on the boundary of the medium. The effect of the refractive index match is predicted correctly by the MC method and by the diffusion approximation. The results demonstrate that matching of the refractive index of the medium significantly improves the contrast and spatial resolution of the spatial photon sensitivity profile (SPSP). The results of simulation of the spatially resolved diffuse reflectance agree well with the results predicted by the diffusion approximation and the experimental results reported earlier.


Subject(s)
Biophysics/methods , Monte Carlo Method , Photons , Scattering, Radiation , Absorption , Diffusion , Light , Models, Biological , Models, Statistical
4.
Nat Struct Biol ; 7 Suppl: 950-2, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11103996

ABSTRACT

Patent protection is available for certain inventions in the field of structural genomics. A review of the patent application procedure is provided, and patentable aspects of protein structural information under US law are discussed. Strategic and international factors to consider when seeking patent protection for an invention also are presented.


Subject(s)
Computational Biology/economics , Computational Biology/legislation & jurisprudence , Databases as Topic/economics , Databases as Topic/legislation & jurisprudence , Patents as Topic/legislation & jurisprudence , Proteins/chemistry , Computational Biology/methods , Genomics/economics , Genomics/legislation & jurisprudence , International Cooperation , Internet , Protein Conformation , Proteins/economics , Proteins/genetics , Proteome/economics , United States
5.
Mol Carcinog ; 29(2): 67-75, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11074603

ABSTRACT

To investigate the role of loss of the p53 tumor suppressor gene in skin carcinogenesis, p53 knockout (p53(-/-)) mice were mated with transgenic mice coexpressing v-Ha-ras, v-fos, or human transforming growth factor alpha (TGFalpha) exclusively in the epidermis by using human keratin 1 (HK1)-based vectors (HK1.ras/fos, HK1.ras/alpha, and HK1.fos/alpha). HK1.ras/fos and HK1.ras/alpha mice displayed epidermal hyperplasia and autonomous benign papillomas to an identical degree between p53(+/+) and p53(+/-) genotypes. However, HK1.ras/fos mice with the p53(-/-) genotype were born with papillomatous skin and died soon after birth. HK1.ras/alpha-p53(-/-) mice also exhibited an increased epidermal hyperplasia, and, similar to HK1.ras/alpha mice with p53(+/+) and p53(+/-) genotypes, these mice rapidly developed spontaneous and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papillomas. These results are in contrast to our previous observation that, HK1.ras, HK1.fos, and HK1.TGFalpha transgenic mice with the p53(-/-) genotype display an unexpected delay in both spontaneous and TPA-promoted papilloma formation compared with mice with p53(+/+) and p53(+/-) genotypes. Taken collectively, our mating experiments between HK1 oncogenic transgenic mice and p53 knockout mice may identify a backup system that effectively compensates for p53 loss. Activation of multiple oncogenes not only partly overcomes such compensation but also synergizes with p53 loss. However, HK1.fos/alpha-p53(-/-) mice failed to exhibit either an increased newborn epidermal hyperplasia or an accelerated spontaneous or TPA-induced papillomas, suggesting that certain combinations of oncogenes, such as with activated Ha-ras, are required for this process. Because neither spontaneous nor TPA-elicited papillomas in p53(-/-) mice progressed to malignancy, additional genetic insults appear to be required for malignant progression.


Subject(s)
Genes, fos/physiology , Genes, p53/physiology , Genes, ras/physiology , Papilloma/genetics , Skin Neoplasms/genetics , Transforming Growth Factor alpha/genetics , Animals , Carcinogens , Epidermis/metabolism , Epidermis/physiology , Female , Gene Expression Regulation , Gene Expression Regulation, Neoplastic , Keratins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Mice, Transgenic , Papilloma/chemically induced , Skin Neoplasms/chemically induced , Tetradecanoylphorbol Acetate
6.
Mol Carcinog ; 27(3): 200-9, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10708482

ABSTRACT

Previously, transgenic mice were generated that overexpressed v-Ha-ras or human transforming growth factor alpha (TGFalpha) exclusively in the epidermis, by means of a targeting vector based on the human keratin 1 gene (HK1). Both transgenics exhibited a similar neonatal phenotype of epidermal hyperplasia/hyperkeratosis and, in adults, spontaneous and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papilloma formation. To assess the synergism in vivo between Ha-ras and TGFalpha, mating experiments were performed. All ras/TGFalpha double genotype progeny (HK1 less than, with dotras/alpha) exhibited an increased epidermal hyperplasia/hyperkeratosis in neonates and accelerated spontaneous papillomatogenesis, compared with single transgenic siblings. HK1 less than, with dotras/alpha mice from the mild lines of HK1 less than, with dotrasxHK1 less than, with dotTGFalpha developed spontaneous papillomas that were not shown in either their parental mice or single transgenic littermates. Unlika in parental or single-genotype siblings, in which TPA promotion-elicited papillomas remained benign, TPA promotion elicited autonomous papillomas in HK1 less than, with dotras/alpha mice and exhibited a novel susceptibility to malignant conversion. Sequence analysis of the endogenous c-Ha-ras from spontaneous and TPA-induced HK1 less than, with dotras/alpha papillomas revealed wild-type sequence. However, carcinomas exhibited c-Ha-ras mutations at codon 61. All tumors analyzed to date expressed wild-type p53. These data provide in vivo evidence that Ha-ras and TGFalpha cooperate in the induction of epidermal hyperplasia and spontaneous tumor formation and predispose to malignant conversion via endogenous c-Ha-ras activation.


Subject(s)
Cell Transformation, Neoplastic/genetics , Epidermal Cells , Genes, ras , Transforming Growth Factor alpha/genetics , Animals , Base Sequence , Cell Division/genetics , DNA Primers , Gene Expression Regulation , Genes, p53 , Mice , Mice, Transgenic , Mutation , Papilloma/genetics , Skin Neoplasms/genetics
7.
Mol Carcinog ; 26(4): 305-11, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10569807

ABSTRACT

To study oncoprotein cooperation in vivo, transgenic mice were established that coexpressed human transforming growth factor-alpha (TGFalpha) and v-fos exclusively in the epidermis by means of a human keratin 1 (HK1)-based vector. HK1.fos/alpha mice exhibited aberrant epidermal proliferation and differentiation and formed spontaneous papillomas that achieved tumor autonomy but did not convert to malignancy. To determine the sensitivity to a chemical promotion stimulus, HK1.fos/alpha mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Previously, after 7 mo TPA promotion of HK1.TGFalpha mice that express moderate levels of TGFalpha elicited papillomas that remained regression-prone and benign for up to 2 yr. In HK1.fos mice, 6 mo TPA elicited papillomas that required spontaneous c-Ha-ras activation and converted to malignancy after 14-16 mo. We now show that in HK1.fos/alpha transgenic genotypes, TPA promotion accelerated papillomatogenesis, with the earliest papilloma appearance at 2 mo after initiation of TPA promotion. These papillomas started to convert to malignancy by 10 mo. Analysis of HK1.fos/alpha papillomas and carcinomas revealed that the endogenous c-Ha-ras gene possessed mutations at codons 12, 13, and 61 at the papilloma stage, but no mutations of the p53 tumor suppressor gene were detected. These data indicate that coexpression of fos and TGFalpha increased epidermal sensitivity to TPA promotion, which accelerated malignant conversion. However, in this transgenic model conversion always required additional genetic events, e.g., activation of the endogenous c-Ha-ras gene.


Subject(s)
Cell Transformation, Neoplastic , Gene Expression Regulation, Neoplastic , Genes, fos , Genes, ras , Papilloma/genetics , Skin Neoplasms/genetics , Tetradecanoylphorbol Acetate/toxicity , Transforming Growth Factor alpha/genetics , Animals , Carcinogens/toxicity , Humans , Mice , Mice, Transgenic , Oncogene Proteins v-fos/genetics , Papilloma/chemically induced , Papilloma/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Skin Neoplasms/chemically induced , Skin Neoplasms/pathology , Transforming Growth Factor alpha/physiology
8.
Mol Carcinog ; 23(3): 185-92, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9833779

ABSTRACT

We previously developed a transgenic mouse model that expresses in the epidermis a murine p53172R-->H mutant (p53m) under the control of a human keratin-1-based vector (HK1.p53m). In contrast to mice with wild-type p53 and p53-knockout mice, HK1.p53m mice exhibit increased susceptibility to chemical carcinogenesis, with greatly accelerated benign papilloma formation, malignant conversion, and metastasis. In the study presented here, we examined the expression pattern of several differentiation markers and observed that p53m tumors exhibited a less differentiated phenotype than tumors elicited in non-transgenic mice. Metastasis in p53m tumors was also associated with a poorly differentiated phenotype. To determine whether genomic instability was associated with a putative gain-of-function role for this p53m, in situ examination of centrosomes was performed in HK1.p53m and equivalent p53-null papillomas. In contrast to HK1.p53m papillomas, which had centrosome abnormalities at high frequencies (75% of cells contained more than three centrosomes/cell), p53-null tumors exhibited few abnormal centrosomes (4% of cells contained more than three centrosomes/cell). To determine whether angiogenesis played a role in the rapid progression of p53m tumors, the expression of vascular endothelial growth factor, a promoter of angiogenesis, and thrombospondin-1, an inhibitor of angiogenesis, was examined in tumors derived from either p53m or p53-knockout mice. Regardless of their p53 status (wild type, p53m, p53-/-), all of the papillomas exhibited similar levels of vascular endothelial growth factor expression and decreased expression of thrombospondin-1 as did normal epidermis. In addition, tumors from different p53 genotypes showed a similar density of blood vessels. Because p53 status did not appear to play an overt role in angiogenesis, these data suggest that p53m accelerates tumorigenesis primarily by exerting a gain of function associated with genomic instability.


Subject(s)
Centrosome/drug effects , Genes, p53/drug effects , Neovascularization, Pathologic/metabolism , Papilloma/blood supply , Papilloma/genetics , Skin Neoplasms/blood supply , Skin Neoplasms/genetics , 9,10-Dimethyl-1,2-benzanthracene , Animals , Carcinogens , Disease Progression , Endothelial Growth Factors/biosynthesis , Humans , Lymphokines/biosynthesis , Mice , Mice, Knockout , Papilloma/chemically induced , Phenotype , Point Mutation , Skin Neoplasms/chemically induced , Tetradecanoylphorbol Acetate , Thrombospondin 1/biosynthesis , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
9.
Oncogene ; 17(1): 35-45, 1998 Jul 09.
Article in English | MEDLINE | ID: mdl-9671312

ABSTRACT

To develop an in vivo model for studying the role of the p53 tumor suppressor in skin carcinogenesis, a murine p53(172H) mutant (equivalent to human p53(175H)) was expressed in the epidermis of transgenic mice, utilizing a targeting vector based on the human keratin 1 gene (HK1.p53m). HK1.p53m mice developed normally and did not exhibit an obvious epidermal phenotype or develop spontaneous tumors. However, these mice demonstrated an increased susceptibility to a two-stage chemical carcinogenesis protocol, with the rate of formation and number of papillomas being dramatically increased as compared to non-transgenic controls. The majority of papillomas in control mice regressed after termination of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, whereas p53m papillomas progressed to carcinomas and metastases. In addition, more advanced malignancy, i.e., undifferentiated spindle cell carcinomas, were exclusively observed in p53m mice. Increased bromodeoxyuridine (BrdU) labeling, accompanied by decreased expression of p21, was observed in HK1.p53m papillomas. In situ examination of centrosomes in HK1.p53m papillomas also revealed marked abnormalities, with 75% of the cells containing > or = 3 centrosomes/cell, whereas centrosome numbers in papillomas from control animals remained normal. These data suggest that the accelerated tumorigenesis observed in chemically-treated p53m mice is most likely due to increased genomic instability resulting from an inhibition of G1 arrest and abnormal amplification of centrosomes.


Subject(s)
Papilloma/genetics , Skin Neoplasms/genetics , Tumor Suppressor Protein p53/physiology , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Bromodeoxyuridine , Carcinogens/toxicity , Centrosome , Disease Susceptibility , Epidermis/pathology , Female , Gene Amplification , Humans , Keratins/genetics , Male , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Papilloma/chemically induced , Papilloma/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras)/genetics , Skin Neoplasms/chemically induced , Skin Neoplasms/pathology , Tetradecanoylphorbol Acetate/toxicity , Transgenes , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics , ras Proteins/biosynthesis
10.
J Invest Dermatol ; 110(6): 867-71, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9620291

ABSTRACT

The efficacy of adenovirus-mediated gene therapy for treatment of metastatic B16 melanomas, established in syngeneic C57BL/6 mice, was assessed via an ex vivo cytokine vaccine approach or via an in vivo strategy utilizing combination cytokine/herpes simplex virus-thymidine kinase (HSV-tk) suicide gene delivery and treatment with ganciclovir (GCV). In the ex vivo tumor vaccine approach, B16 melanoma cells, transduced in vitro by adenovirus containing either interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha cytokine genes and gamma irradiated, were subcutaneously injected into the flank and a distant subcutaneous challenge injection of unmodified B16 melanoma cells was performed 15 d later. Significant reductions in challenge tumor volume were observed in the IL-2 group (75% reduction; p = 0.02) and in the GM-CSF group (88% reduction; p = 0.0006), whereas the effect for tumor necrosis factor-alpha was not statistically significant. In the in vivo treatment of established melanomas, this cytokine approach was combined with a suicide gene therapy and subcutaneous B16 melanomas were directly injected with (i) IL-2/recombinant, replication-deficient adenovirus (adv) and thymidine kinase (tk)/adv, (ii) GM-CSF/adv, IL-2/adv, and tk/adv, or (iii) control beta-galactosidase (beta-gal)/adv and tk/adv. After intraperitoneal application of GCV (10 mg per kg) for 6 d, the residual tumor masses were excised and the animals challenged with unmodified B16 cells. Challenge tumor growth was reduced by 56% for the IL-2/tk/adv/GCV treatment (p = 0.041) and by 77% for the GM-CSF/IL-2/tk/adv/GCV treatment p (p = 0.037), in comparison with the beta-gal/tk/GCV control group. These data may hold significant promise for the development of effective ex vivo and in vivo gene therapy modalities to counter the highly metastatic nature of human melanoma.


Subject(s)
Adenoviridae/genetics , Antineoplastic Agents/metabolism , Genetic Therapy , Melanoma, Experimental/therapy , Animals , Antineoplastic Agents/immunology , Antineoplastic Agents/therapeutic use , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Disease Models, Animal , Female , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Interleukin-2/genetics , Interleukin-2/immunology , Interleukin-2/therapeutic use , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Mutagenesis, Insertional , Neoplasm Metastasis/genetics , Neoplasm Metastasis/immunology , Neoplasm Metastasis/prevention & control , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Tumor Cells, Cultured
12.
Proc Natl Acad Sci U S A ; 94(6): 2386-91, 1997 Mar 18.
Article in English | MEDLINE | ID: mdl-9122204

ABSTRACT

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.


Subject(s)
Epidermis/physiology , Keratinocytes/cytology , Keratinocytes/physiology , Receptors, Transforming Growth Factor beta/biosynthesis , Skin Aging , Skin Physiological Phenomena , Transforming Growth Factor beta/pharmacology , Amino Acid Sequence , Animals , Animals, Newborn , Cell Division/drug effects , Cells, Cultured , Epidermis/drug effects , Epidermis/pathology , Epitopes/chemistry , Female , Genes, myc , Humans , Hypertrophy , Keratinocytes/drug effects , Keratosis/genetics , Male , Mice , Mice, Inbred ICR , Mice, Inbred Strains , Mice, Transgenic , Mitotic Index , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-myc/biosynthesis , Receptor, Transforming Growth Factor-beta Type II , Skin/pathology
13.
Cancer Res ; 56(19): 4413-23, 1996 Oct 01.
Article in English | MEDLINE | ID: mdl-8813135

ABSTRACT

To investigate the effect of p53 tumor suppressor gene loss in the mouse skin model of multistage carcinogenesis, p53 knockout mice, generated by gene targeting (p53 -/-), were mated to transgenic mice expressing v-rasHa (HK1.ras), v-fos (HK1.fos), or human transforming growth factor alpha+HK1.TGFalpha) exclusively in the epidermis, by means of a keratin K1-based targeting vector (HK1). HK1-p53 transgenic progeny expressing wild-type p53 alleles (p53 +/+) or hemizygous for the p53 knockout allele (p53+/-) were identical to parental HK1 lines and exhibited neonatal epidermal hyperplasia or wound-associated hyperplasia in adults, together with spontaneous or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced benign papillomas. Mating to p53-/- did not lead to the expected tumorigenesis in adults. Instead, whereas HK1.ras or HK1.TGFalpha transgenic mice null for p53 (HK1.ras-p53-/- and HK1.TGFalpha-p53-/-, respectively) retained the neonatal epidermal hyperplasia phenotype, in adults, spontaneous and TPA-promoted papilloma formation was blocked. Similarly, wound-associated epidermal hyperplasia/hyperkeratosis, a hallmark of adult HK1.fos phenotypes, was completely absent in HK1.fos-p53 -/- mice. Histological, immunofluorescence, and bromodeoxyuridine labeling analysis of neonatal or adult epidermis in HK1-p53 transgenic genotypes +/+, +/-, and -/- for p53 revealed no obvious differences in morphology, expression of keratinocyte differentiation markers, or mitotic index attributed to p53 loss. To determine whether the paradoxical absence of papillomas centered on up-regulation of p53 target genes, WAF1/CIP1/p21 RNA expression levels were examined in TPA promotion experiments. WAF1/CIP1/p21 expression increased in response to TPA promotion in all HK1-p53 transgenic genotypes regardless of p53 status. However, in HK1-p53 null genotypes, although TPA-induced, p53-independent WAF1/CIP1/p21 expression was observed, no large increase in expression was associated with the observed paradoxical tumorigenesis block. These data suggest that epidermis is somewhat resistant to the neoplastic effects of p53 loss, possibly possessing several compensatory systems. Alternatively, there may be a requirement forp53 expression in response to TPA or a wound-promotion stimulus in mouse epidermis.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cocarcinogenesis , Epidermis/metabolism , Genes, p53 , Oncogene Protein p21(ras)/biosynthesis , Oncogene Proteins v-fos/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Transforming Growth Factor alpha/biosynthesis , Animals , Animals, Newborn , Cell Differentiation/drug effects , Cell Division/drug effects , Crosses, Genetic , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Cyclins/genetics , Epidermis/pathology , Genes, fos , Genes, ras , Hyperplasia , Keratinocytes/metabolism , Keratinocytes/pathology , Mice , Mice, Knockout , Mice, Transgenic , Oncogene Protein p21(ras)/genetics , Oncogene Proteins v-fos/genetics , Recombinant Fusion Proteins/genetics , Tetradecanoylphorbol Acetate/toxicity , Transforming Growth Factor alpha/genetics , Transgenes
14.
J Invest Dermatol ; 106(6): 1163-8, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8752651

ABSTRACT

To assess the efficacy of an in vivo adenoviral-mediated cytotoxic gene therapy, human melanomas were established in nude mice and transduced with herpes simplex virus-thymidine kinase (tk) followed by treatment with ganciclovir (GCV). In initial experiments, adenovirus (adv) containing the beta-galactosidase reporter gene was employed to determine melanoma cell infectivity in vitro. In comparison to murine melanoma cell lines B16 and K1735-M2, human A375-SM cells exhibited up to a 10-fold greater susceptibility to adenoviral transduction, similar to the degree of infectivity found for human epidermal HaCaT cells. In addition, human A375-SM melanoma cells exhibited a greater sensitivity in vitro to the cytotoxic effects of transduction with tk-adv and treatment with GCV, which was mediated by a strong bystander effect. In vivo, intratumoral injection of relatively large human melanomas (160 mm3) with 1.2 X 109 pfu of tk-adv, followed by intraperitoneal GCV treatment (60 mg/kg twice daily) over 4 days, typically resulted in a 50% reduction in melanoma growth rate compared to mock or untreated controls. Moreover, histometrical analysis employing a rigorous computerized imaging system revealed that the residual viable tumor area in the tk-adv/GCV-treated group was only one-fifth that of solvent controls. These data show that adv is a highly efficient in vivo gene delivery system to treat experimental human melanomas. In comparison to a previous murine melanoma study, human melanomas appeared to exhibit a greater sensitivity to this cytotoxic treatment in vivo, which may hold significant promise for development of effective gene therapy modalities to treat melanoma in humans.


Subject(s)
Adenoviridae , Gene Transfer Techniques , Genetic Vectors , Melanoma/therapy , Simplexvirus/enzymology , Thymidine Kinase/genetics , Adenoviridae/genetics , Animals , Cell Division/drug effects , Ganciclovir/therapeutic use , Genes, Reporter , Humans , Injections, Intraperitoneal , Melanoma/drug therapy , Melanoma/pathology , Mice , Mice, Nude , Transduction, Genetic , Tumor Cells, Cultured , beta-Galactosidase/genetics
15.
J Investig Dermatol Symp Proc ; 1(2): 162-76, 1996 Apr.
Article in English | MEDLINE | ID: mdl-9627712

ABSTRACT

Skin cancer is one of the most prevalent forms of human neoplasia with a frequency approaching that of all other neoplasms combined. Given this alarming statistic, which may be further exacerbated by increased ultraviolet B irradiation from ozone depletion, it is vital that realistic, relevant model systems are developed to increase our understanding of the underlying molecular mechanisms of carcinogenesis that result in or evaluate new treatment modalities. Toward this goal, the ability to stably introduce genes into the germline of mice has greatly enhanced prospects for generation of transgenic animal models of multistage molecular carcinogenesis. Moreover, when genes are combined with regulatory sequences that target their expression to specific tissues, investigators are able to study neoplasia both in the context of living organisms and in the tissues suspected of being the targets of these genes. The epidermis is an attractive tissue for targeted gene expression; not only is it a model for epithelial diseases in general, but the accessibility of the epidermis allows easy detection of progressive pathological changes that result from transgene expression and facilitates assessment of the potential role played by environmental factors. We have developed a targeting vector based on the human keratin gene (HK1), which is expressed exclusively in the epidermis of transgenic mice, at a late stage in development and in both basal and differentiated cells. Through the use of this targeting ability, rasHa, fos, and TGF alpha transgenic mice have been developed that exhibit preneoplastic epidermal hyperplasia and hyperkeratosis, and later benign, regression prone papillomas. Together, coexpression of two oncogenes cooperated to give autonomous papillomas, which possessed the phenotypic stability to allow assessment of a third genetic event, namely loss of the p53 tumor suppressor gene, via mating with p53 knockout mice. Loss of p53 expression, however, identified a paradoxical block of papillomatogenesis. This latter result suggests that the epidermis possesses several mechanisms that can effectively compensate for the loss of important tumor suppressor functions and may have evolved to render the skin relatively resistant to the effects of environmental carcinogens.


Subject(s)
Skin Neoplasms/etiology , Animals , Genes, fos/physiology , Genes, p53/physiology , Genes, ras/physiology , Humans , Mice , Mice, Transgenic , Transforming Growth Factor alpha/physiology
17.
Cell Growth Differ ; 6(5): 579-86, 1995 May.
Article in English | MEDLINE | ID: mdl-7544154

ABSTRACT

Transgenic mice that expressed v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos) developed preneoplastic epidermal hyperplasia and hyperkeratosis after long latency and an associated wound promotion stimulus. To assess the requirements for papilloma formation and malignant conversion and determine the sensitivity to a chemical promotion stimulus, HK1.fos mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). HK1.fos mice were sensitive to TPA promotion but developed papillomas only after long latency (20-30 weeks of promotion) and in relatively few numbers per animal, suggesting the necessity of an additional genetic event prior to overt lesion formation. Consistent with this idea, at 60 weeks, on cessation of TPA promotion, these HK1.fos TPA-papillomas were found to be autonomous, TPA-independent tumors which persisted, grew larger, and converted to malignancy. Analysis of HK1.fos tumor RNA and DNA identified endogenous c-rasHa mutations at codons 12 and 61 in papillomas and carcinomas; however, no p53 tumor suppressor gene mutations were detected. These data indicate that epidermal expression of v-fos induces sensitivity to TPA promotion, but since additional genetic events, such as endogenous c-rasHa activation, appear to be required in tumorigenesis, v-fos may predominantly play a role in the mechanism of promotion to achieve papilloma autonomy and TPA independence. Furthermore, spontaneous malignant conversion in this model does not appear to involve mutations in the p53 tumor suppressor gene.


Subject(s)
Cell Transformation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, fos , Genes, p53/genetics , Genes, ras/genetics , Mutation , Tetradecanoylphorbol Acetate/pharmacology , Animals , Base Sequence , Biomarkers , Carcinoma/genetics , Carcinoma/pathology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , DNA, Neoplasm , Epidermis/pathology , Hyperplasia/genetics , Hyperplasia/pathology , Keratins/analysis , Keratosis/genetics , Keratosis/pathology , Mice , Mice, Transgenic , Molecular Sequence Data , Oncogene Proteins v-fos/biosynthesis , Oncogene Proteins v-fos/genetics , Papilloma/genetics , Papilloma/pathology , RNA, Neoplasm , ras Proteins/biosynthesis , ras Proteins/genetics
18.
J Invest Dermatol ; 104(3): 313-7, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7860993

ABSTRACT

To assess the potential of an in vivo, adenovirus-mediated gene therapy approach for the treatment of malignant melanoma, the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-Ek) transfer and administration of ganciclovir (GCV) was investigated using a nude mouse model. Initially, B16 murine melanoma cells were efficiently transduced in vitro by a recombinant replication-defective adenovirus containing the HSV-tk gene (ADV/RSVtk), and rendered sensitive to cell killing by 10 micrograms/ml GCV. A significant "bystander effect" was observed at low multiplicity of infection in comparison of cell killing to control B16 transduction by adenovirus containing the beta-galactosidase gene (ADV/RSV-beta-gal). In vivo, melanomas established from subcutaneous injection of 4 x 10(5) B16 cells were injected after 14 d with 1 x 10(10) ADV/RSV-tk viral particles. Subsequent treatment for 6 d with GCV resulted in an inhibition of melanoma growth, with an approximately 40-50% reduction in melanoma volume in comparison to controls in repeated experiments. These data demonstrate that adenovirus-mediated gene transfer can function as an efficient delivery system to reduce established tumor burden in vivo. This result may hold significant promise for the development of effective in situ gene therapy for melanoma in humans.


Subject(s)
Adenoviridae/genetics , Genetic Therapy , Genetic Vectors , Melanoma, Experimental/prevention & control , Simplexvirus/genetics , Thymidine Kinase/genetics , Animals , Ganciclovir/therapeutic use , Gene Transfer Techniques , Melanoma, Experimental/genetics , Mice , Transduction, Genetic , Tumor Cells, Cultured
19.
Nat Struct Biol ; 2(2): 139-43, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7749918

ABSTRACT

Assembly of proteins within lipid bilayers is essential for the biogenesis and function of biological membranes. Little is known, however, about the underlying mechanism of assembly, and it is not clear whether it is possible to observe individual folding steps for integral membrane proteins either in vivo or in vitro. Fluorescence spectroscopy is used here to follow the time course of folding events for bacteriorhodopsin in mixed detergent/lipid micelles. Transient folding-intermediates are detected and binding of the retinal chromophore occurs at a late stage, when it binds to an apoprotein intermediate.


Subject(s)
Bacteriorhodopsins/chemistry , Membrane Proteins/chemistry , Protein Folding , Apoproteins/metabolism , Bacteriorhodopsins/metabolism , Cholic Acids , Detergents , Dimyristoylphosphatidylcholine , Micelles , Models, Molecular , Protein Binding , Retinaldehyde/metabolism , Schiff Bases , Sodium Dodecyl Sulfate , Spectrometry, Fluorescence
20.
J Invest Dermatol ; 104(1): 58-61, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7528246

ABSTRACT

The immortalized human keratinocyte cell line HaCaT was used to assess the effect of interferon-gamma (IFN-gamma) on expression of keratin K17. Both IFN-gamma and K17 have been implicated in the pathophysiology of psoriasis. Western and quantitative enzyme-linked immunosorbent assay analyses demonstrated increasing induction of K17 protein by 48 h exposure to IFN-gamma at concentrations of 10, 50, and 250 U/ml. At 50 U/ml IFN-gamma, immunohistochemical analysis revealed numerous K17-positive foci, whereas in situ hybridization demonstrated K17 message in the majority of cells. In addition, at low (5 U/ml) concentrations of IFN-gamma, cell proliferation and protein synthesis decreased, as determined by 3H-thymidine labeling and 14C-amino acid uptake. These data suggest that aberrant K17 expression observed in psoriatic lesions may be a consequence of IFN-gamma overexpression, and that the HaCaT cell line may be a useful in vitro model system to elucidate the underlying mechanisms.


Subject(s)
Interferon-gamma/pharmacology , Keratinocytes/chemistry , Keratins/physiology , Antigens, Differentiation/analysis , Blotting, Western , Cell Line , Enzyme-Linked Immunosorbent Assay , Humans , Keratins/drug effects , Keratins/genetics , Psoriasis/diagnosis , RNA, Messenger/analysis , Up-Regulation
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