Alternative genetic pathways in colorectal carcinogenesis

The comparative typing of matched tumor and blood DNAs at dinucleotide repeat (microsatellite) loci has revealed in tumor DNA the presence of alleles that are not observed in normal DNA. The occurrence of these additional alleles is possibly due to replication errors (RERs). Although this observation has led to the recognition of a subtype of colorectal cancer with a high incidence of RERs (caused by a deficiency in DNA mismatch repair), a thorough analysis of the RER frequency in a consecutive series of colorectal cancers had not been reported. It is shown here that the extensive typing of 88 colorectal tumors reveals a bimodal distribution for the frequency of RER at microsatellite loci. Within the major mode (75 tumors, RER− subtype), the probability that a locus exhibited instability did not differ significantly among loci and tumors, being 0.02. The subsequent development of a statistical test for an operational discrimination between the RER− and RER+ subtypes indicated that the probability of misclassification did not exceed 0.001 in this series. The frequency of K-ras mutation was found to be equivalent in the two subtypes. However, in the RER+ tumors, the p53 gene mutation was less frequently detected, the adenomatous polyposis coli (APC) mutation was rare, and the biallelic inactivation of either of these genes was not observed. Furthermore, the concomitant occurrence of APC and tumor growth factor β receptor type II gene alterations was found only once. These data suggest that the repertoires of genes that are frequently altered in RER+ and RER− tumors may be more different than previously thought.

Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups

Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular “sensor” molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the β-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

L-selectin can facilitate metastasis to lymph nodes in a transgenic mouse model of carcinogenesis

L-selectin mediates homing of lymphocytes to lymph nodes (LN). Transgenic mice that express rat insulin promoter regulated simian virus 40 Tag (RIP-Tag) develop large, local cancers that metastasize to liver but not LN. To test whether this lack of LN metastases reflects their absence from the circulation, transgenic mice were produced that express Tag (T), L-selectin (L), and Escherichia coli LacZ (Z), in pancreatic β cells. LTZ mice developed insulinomas that specifically had LN metastases; metastasis was blocked by an anti L-selectin mAb. LacZ+ tumor cells from these LN homed to secondary LN upon transfer. These results suggest that the highly vascularized islet carcinomas are shedding tumor cells into the bloodstream, which is a necessary but insufficient condition for metastasis to occur; L-selectin can facilitate homing of such tumor cells to LN, resulting in metastasis.

Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis.

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice.

High-risk human papillomaviruses (HPVs), including type 16, have been identified as factors in cervical carcinogenesis. However, the presence and expression of the virus per se appear to be insufficient for carcinogenesis. Rather, cofactors most likely are necessary in addition to viral gene expression to initiate neoplasia. One candidate cofactor is prolonged exposure to sex hormones. To examine the possible effects of estrogen on HPV-associated neoplasia, we treated transgenic mice expressing the oncogenes of HPV16 under control of the human keratin-14 promoter (K14-HPV16 transgenic mice) and nontransgenic control mice with slow release pellets of 17beta-estradiol. Squamous carcinomas developed in a multistage pathway exclusively in the vagina and cervix of K14-HPV16 transgenic mice. Estrogen-induced carcinogenesis was accompanied by an incremental increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-HPV16 mice. Expression of the HPV transgenes in untreated transgenic mice was detectable only during estrus; estrogen treatment resulted in transgene expression that was persistent but not further upregulated, remaining at low levels at all stages of carcinogenesis. The data demonstrate a novel mechanism of synergistic cooperation between chronic estrogen exposure and the oncogenes of HPV16 that coordinates squamous carcinogenesis in the female reproductive tract of K14-HPV16 transgenic mice.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Hormones and mammary carcinogenesis in mice, rats, and humans: a unifying hypothesis.

An attempt has been made to put forward a unifying hypothesis explaining the role hormones play in the genesis of mammary cancers of different phenotypes and genotypes in mice, rats, and humans. Most mammary cancers in these species originate in luminal mammary epithelial cells lining the mammary ducts and alveoli. These cancers are histopathologically diverse and are classified on the basis of growth requirements as hormone-dependent or hormone-independent tumors. In most strains of mice, mammary cancers at the time of detection are largely of the hormone-independent type; in rats, almost all mammary cancers are hormone-dependent, while humans have both phenotypes. In spite of these differences, in vivo studies show that hormones (ovarian and pituitary) are essential for luminal mammary epithelial cell proliferation and also for the development of mammary cancers of both hormone-independent and hormone-dependent types. This article, based on our extensive in vivo and in vivo studies and on current literature, proposes a model to explain the central role of hormones in the genesis of all types of mammary cancers. The model attempts to address the following questions: (i) how hormones regulate luminal mammary epithelial cell proliferation, (ii) why hormones are required for the genesis of mammary cancers of all phenotypes and genotypes, including those which are always classified as hormone-independent tumors, and (iii) why the three species (mouse, rat, and human) have consistently different ratios of hormone-dependent to hormone-independent tumors.

Epidemiological aspects of carcinogenesis,

Proceedings of a workshop conducted by the Interdisciplinary Communications Program of the Smithsonian Institution, held at the Belmont Conference Center, Elkridge, Md., Oct. 22-25, 1972

Molecular introduction to head and neck cancer (HNSCC) carcinogenesis

Of all human cancers, HNSCC is the most distressing affecting pain, disfigurement, speech and the basic survival functions of breathing and swallowing. Mortality rates have not significantly changed in the last 40 years despite advances in radiotherapy and surgical treatment. Molecular markers are currently being identified that can determine prognosis preoperatively by routine tumour biopsy Leading to improved management of HNSCC patients. The approach could help decide which early stage patient should have adjuvant neck dissection and radiotherapy, and whether Later stage patients with operable lesions would benefit from resection and reconstructive surgery or adopt a conservative approach to patients with poor prognosis regardless of treatment. In the future, understanding these basic genetic changes in HNSCC would be important for the management of HNSCC. (C) 2004 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.