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TitleTransformation of Human Epithelial Cells (1992) : Molecular and Oncogenetic Mechanisms
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Table of Contents
Title Page
Copyright Page
Chapter 1 In Vitro Cellular Aging and Immortalization
Chapter 2 Detection of Growth Factor Effects and Expression in Normal and Neoplastic Human Bronchial Epithelial Cells
Chapter 3 Human Cell Metabolism and DNA Adduction of Polycyclic Aromatic Hydrocarbons
Chapter 4 Human Esophageal Epithelial Cells: Immortalization and In Vitro Transformation
Chapter 5 Transformation of Human Endometrial Stromal Cells In Vitro
Chapter 6 Factors Influencing Growth and Differentiation of Normal and Transformed Human Mammary Epithelial Cells in Culture
Chapter 7 Transformation of Colon Epithelial Cells
Chapter 8 Multistep Carcinogenesis and Human Epithelial Cells
Chapter 9 Morphologic and Molecular Characterizations of Plastic Tumor Cell Phenotypes
Chapter 10 Oncogene and Tumor Suppressor Gene Involvement in Human Lung Carcinogenesis
Chapter 11 Events of Tumor Progression Associated with Carcinogen Treatment of Epithelial and Fibroblast Compared with Mutagenic Events
Chapter 12 Progression from Pigment Cell Patterns to Melanom as in Platyfish-Swordtail Hybrids — Multiple Genetic Changes and a Theme for Tumorigenesis
Document Text Contents
Page 2


Human Epithelial Cells:

Molecular and Oncogenetic


George E. Milo, M.S., Ph.D.
Professor, Department of Medical Biochemistry

Director of Carcinogenesis and Molecular Toxicology
Comprehensive Cancer Center

The Ohio State University
Columbus, Ohio

Bruce C. Casto, M.S., Sc.D.
Director of Research

Environmental Health and Research Testing, Inc.
Research Triangle Park, North Carolina

Charles F. Shuler, D.M.D., Ph.D.
Assistant Professor, Center for Craniofacial Molecular Biology

University of Southern California
Los Angeles, California

\CP* J Taylor & Francis Group
^***^/ Boca Raton London New York

CRC Press is an imprint of the
Taylor & Francis Group, an informa business

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150 Transformation of Human Epithelial Cells

protein.119"121 Introduction of p53 into primary cells was reported to result in
immortalization and, in cooperation with the ras oncogene, transformation
of the cells, although these alterations are now attributed to mutant forms of

Studies on human colorectal tumors have provided strong evidence to
link abnormalities in the p53 gene and tumorigenesis.31'113 As mentioned
above, almost 75% of the colorectal cancers exhibit a complete loss of one
of the two p53 alleles.31 The second allele in the two tumors studied in detail
was mutated. The mutations involved a substitution of alanine for valine at
codon 143 of one tumor and a histidine for arginine at codon 175 of the
second tumor. Most interestingly, the mutations occurred in this highly con-
served protein domain. Mutations in these locations have previously been
reported to greatly alter the biological properties of murine p53.116-122'123 These
results strongly implicate p53 mutations in colon carcinogenesis, although
the associated mechanisms are a matter of speculation. It is possible that the
normal p53 gene products interact with specific DNA and/or proteins to cause
suppression of colon epithelial cell proliferation and neoplastic growth. Mu-
tations in p53 genes may result in products that may prevent interaction with
specific macromolecules or compete with normal p53 proteins to act in a
negative dominant manner. Further, the effects of the mutated gene products
may be more pronounced when the normal allele is lost, as occurs in colorectal
tumors (loss of 17p). It is interesting to note that wild-type p53 was shown
to stimulate transcription, whereas the mutated forms were unable to act as
transcriptional activators.124-125 Therefore, it was suggested that the inability
of p53 mutant proteins to induce transcription may cause the transformation.
Another possibility to consider is the interaction between ras oncogene and
p53. There are reports that mutant mouse p53 genes can cooperate with ras
oncogenes to transform rodent cells.126-127 Ras oncogenes are highly expressed
in approximately 50% of colorectal adenomas. The simultaneous occurrence
of mutations in ras oncogenes and p53, which are present in colorectal lesions,
presents intriguing possibilities.

Another allelic loss in colorectal tumors occurs in chromosome 18q. Such
deletions have been detected in approximately 70% of the carcinomas and
50% of late adenomas.24'29 As noted above, chromosome deletions frequently
indicate the presence of tumor suppressor genes. Recently, a candidate gene,
termed DCC, has been identified as a possible tumor suppressor gene in
chromosome 18q.29 This gene apparently encodes a protein with amino acid
sequences similar to neural cell adhesion molecules and is related to plasma
membrane glycoproteins. The identification of the gene was based on several
parameters:29 (1) one allele of the DCC gene was found deleted in 71% of
the colorectal neoplasms, (2) the DCC gene was expressed in all normal
mucosal tissues, but its expression was greatly reduced or absent in 88% of
colon tumor cell lines, and (3) somatic mutations of the DCC gene occurred
in almost 13% of the carcinomas. The mechanisms by which the DCC gene is
involved in colorectal neoplasms is not understood; reduction in its expression

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Chopra 151

could reduce the growth-inhibiting properties and other cell surface properties.
Much evidence exists to suggest that neoplastic transformation involves cell
surface alterations. For instance, loss of contact inhibition of growth in vitro
is considered an important phenotype of preneoplastic and/or neoplastic trans-
formation; malignant transformation is believed to involve cell-cell and cell-
basement membrane interactions. Further studies, however, are necessary to
define the role of the DCC gene in colorectal carcinoma.

Besides genetic modifications (deletions/mutations) in protooncogenes
and tumor suppressor genes, abnormal expression of oncogenes caused by
gene amplification, translocation, or rearrangements may contribute to tu-
morigenesis. Altered expression of oncogenes, other than those discussed
above, have been reported in colorectal carcinomas. Among these, enhanced
expression of c-myc oncogene in the adenomas and colon carcinomas relative
to the normal mucosa have been observed.27-128-131 No structural alterations
of c-myc oncogene, however, have been reported. The c-myc encodes a protein
with a 62-kDa molecular weight which is predominantly located in the nucleus
and is believed to be involved in cellular proliferation and differentiation.
Although the precise function of c-myc protein is not known, the cellular
homologs of the gene are highly preserved throughout the species, indicating
its considerable importance. Most cells have some expression of c-myc, but
it is generally elevated when the cells are stimulated to divide and decreases
in terminally differentiated cells. In the normal colon mucosa, c-myc expres-
sion was positive in the middle crypt zone and surface cells, while the basal
crypts were essentially negative.129 132 The staining was mainly cytoplasmic,131

but another study reported it to be predominantly nuclear.128 In adenomatous
polyps, the expression was elevated, the staining being predominant in areas
of dysplasia.131 All colorectal tumors also exhibited higher levels of c-myc
expression, which appeared unrelated to the clinical behavior or degree of
differentiation of the tumors.128'130 Well-differentiated tumors, however, showed
more cytoplasmic staining than the poorly differentiated.131 In another study,
however, a direct correlation was observed between the expression of c-myc
protein and mRNA and degree of differentiation of colorectal tumors.129 Well-
differentiated tumors exhibited higher expression relative to the poorly dif-
ferentiated. Nevertheless, taken collectively, these studies clearly indicate
that c-myc oncogenes have an important role in the production of colorectal
carcinoma, although the mechanisms involved remain unclear. Other oncogenes
with enhanced expression in colon tumors include c-erbE-2 and c-/nv6.27'I33>134

Hypomethylation of DNA has also been reported in colon tumors.135-136 The
loss of the methyl groups might cause alterations in the chromosomes and
subsequent genetic instability.137

Studies on human colon tumorigenesis have identified important genetic
events which may be associated with the specific stages of the multistep
tumorigenesis. Four alterations, i.e., mutations in the c-ras genes and allelic
deletions in chromosomes 5q, 17p, and 18q, are predominant. Ras gene
mutations and allelic deletions in 5q occur predominantly during the early

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312 Transformation of Human Epithelial Cells

benzamide effect, 215-216, 218-219,
221, 231

in situ hybridization, 216
isolation and growth, 214
methylmethane sulfonate effect, 215,

217-218, 219
molecular characterization, 223-230
northern blot analysis, 217
phenotype conversion, 212-213, 217,

218, 219-223, 230-231
polymerase chain reaction, 216-217,

228, 229
tumorigenicity, 215

genomic mutations, 225, 228, 229, 230
growth on soft agar, 265, 267
keratinocytes, 173, 175-176, 178, 179
keratins of. 69
loss of heterozygosity, 241-243

growth on soft agar, 265, 267
invasiveness, 268
tumorigenicity, 268, 275-276
vs. initiated characteristics, 262

surface characterization, 274
tumorigenicity, 215, 220, 221, 222, 223

Sr factor, 288, 293
src oncogenes, 183-185
SS (Sjogren's syndrome), 196
Stress fibers, 95-97
Stromal cells, see Endometrial stromal

Sulfotransferase, 34
SV40, see Simian virus-40
Swordtail, see Xiphorophorus
Synchronous fluorescence spectroscopy, 49
Syrian hamster embryo (SHE) cells, 96

T-antigen, see Simian virus-40
T4 polynucleotide kinase, 50, 52, 270
T47D cell line, see Human mammary epi-

thelial cells, carcinoma cell lines
TBAB (tetrabutylammonium bromide), 38,

Temperature shift, 90-93, 108, see also

Simian virus-40; Endometrial
stromal cells

Tetrabutylammonium bromide (TBAB),
38, 39

Tetraols, see Benzo(a)pyrene, DNA

12-0-Tetradecanoyl-phorbol-13-acetate, 88,

TGF-a (transforming growth factor a),
14-15, 17, 18, 21-23, see also
Growth factors

TGF-p (transforming growth factor (3),
75-76, 79, 80, 127-128, 133-134,
see also Growth factors

Thin-layer chromatography, 36, 50-51,

5-Thioguanine, 276-277
TPA (12-O-tetradecanoyl-phorbol-13-

acetate), 88, 93
Transfection, see individual entries
Transformation suppressor genes, 109
Transforming growth factor a (TGF-a),

14-15, 17, 18, 21-23, see also
Growth factors

Transforming growth factor 0 (TGF-(3),
75-76, 79, 80, 127-128, 133-134,
see also Growth factors

Tritium, 35, 37
Trypsin, 7
tsSV40, see Simian virus-40
Tu gene, 298
Tumor cells, plastic, see Squamous cell

carcinoma, anchorage-independent
cell line

Tumor suppression hypothesis, 148-152
Tumor suppressor genes, 96, 109, 142,

145, 148-152, 240-246, see also
Melanoma suppressor gene

Tumor viruses, see individual entries
Tumorigenesis, genetic basis of, 145-152,

Tumorigenicity, see also individual entries

assay, 190-191
human chromosomes, 6. 148-152
monochromosome fusion technique,

NHE transformed cells, 76, 78, 79
squamous cell carcinoma cell lines, 215,

220, 221, 222, 223, 275-276
suppression in somatic cell hybrids, 244,

Tumors, see also individual entries

formation in mouse vs. human, 2
growth factor production in, 15-16
induction by carcinogen-transformed

populations, 275-276
phenotypical characterization, 212, 213
promoters, 88-90

Tyrosine kinase, 108

Page 326

Index 313


UDP-glucuronosyltransferase, 34
Unscheduled DNA synthesis (UDS),

Unsymmetrical dimethyhydrazine

(UDMH), 271, 273-274


czs-Vicinyl hydroxyls, 44-45, 54
Vimentin, 73-74, 124, 125, 127

Wilms' tumor, 241, 244

Xiphorophorus spp., 286-288, see also

Xmrk oncogene, 297

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