Alfred G. Knudson

Two genetic hits (more or less) to cancer.

Most cancers have many chromosomal abnormalities, both in number and in structure, whereas some show only a single aberration. In the era before molecular biology, cancer researchers, studying both human and animal cancers, proposed that a small number of events was needed for carcinogenesis. Evidence from the recent molecular era also indicates that cancers can arise from small numbers of events that affect common cell birth and death processes.

Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer.

This article extends our previous quantitative analysis of the relationship between the dynamics of the primary structure of DNA and mutagenesis associated with single-strand lesions to an analysis of the production and processing of endogenous double-strand breaks (EDSBs) and to their implications for oncogenesis. We estimate that in normal human cells ≈1% of single-strand lesions are converted to ≈50 EDSBs per cell per cell cycle. This number is similar to that for EDSBs produced by 1.5–2.0 Gy of sparsely ionizing radiation. Although EDSBs are usually repaired with high fidelity, errors in their repair contribute significantly to the rate of cancer in humans. The doubling dose for induced DSBs is similar to doubling doses for mutation and for the induction of carcinomas by ionizing radiation. We conclude that rates of production of EDSBs and of ensuing spontaneous mitotic recombination events can account for a substantial fraction of the earliest oncogenic events in human carcinomas.

A continuum model for tumour suppression

This year, 2011, marks the forty-year anniversary of the statistical analysis of retinoblastoma that provided the first evidence that tumorigenesis can be initiated by as few as two mutations. This work provided the foundation for the two-hit hypothesis that explained the role of recessive tumour suppressor genes (TSGs) in dominantly inherited cancer susceptibility syndromes. However, four decades later, it is now known that even partial inactivation of tumour suppressors can critically contribute to tumorigenesis. Here we analyse this evidence and propose a continuum model of TSG function to explain the full range of TSG mutations found in cancer.

Regression of Neuroblastoma IV-S: A Genetic Hypothesis

Neuroblastoma is the classic tumor that illustrates the phenomenon of spontaneous regression. The most dramatic cases of regression occur in those infants who have disseminated disease involving li...

Mechanism and relevance of ploidy in neuroblastoma

Neuroblastoma has a broad spectrum of clinical behavior, ranging from spontaneous regression to dissemination and fatality. The heterogeneity that has long puzzled many investigators has been shown by more recent studies to be closely correlated with various clinical and genetic factors. Tumor cell ploidy is one of the factors; diploid and near‐triploid neuroblastomas show poor and excellent clinical outcomes, respectively. We offer a hypothesis that explains how the ploidy state of the tumor plays a fundamental role in this heterogeneity, and why various prognostic factors are correlated with each other. This hypothesis may be applicable to tumors other than neuroblastoma.

Radiation dose-rate effects, endogenous DNA damage, and signaling resonance

We previously concluded, from our analysis of the published data of other investigators, that the yield of germ-line and somatic mutations after exposure to ionizing radiation is parabolically related to the logarithm of the dose-rate at which a given dose is administered. Here we show that other data reveal a similarly parabolic relationship for other ionizing radiation-associated phenomena, namely, genetic recombination, chromosomal translocation, cell inactivation and lethality, and human leukemogenesis. Furthermore, the minima for all effects fall in a relatively narrow range of the dose-rate logarithms. Because the only mechanism common to all of these phenomena is the double-strand break (DSB) in DNA, we refer to our previous analysis of the endogenous production of DSBs, from which we concluded that ≈50 endogenous DSBs occur per cell cycle, although most are repaired without error. Comparison then reveals that their rate of production falls within the range of minima for the several end points pursuant to radiation-induced DSBs. We conclude that the results reflect a physiological principle whereby signals originating from induced DSBs elicit responses of maximal effectiveness when they are produced at a rate near that of the production of endogenous DSBs. We refer to this principle as “signaling resonance.”

Altered Gene Expression in Morphologically Normal Epithelial Cells from Heterozygous Carriers of BRCA1 or BRCA2 Mutations

We hypothesized that cells bearing a single inherited “hit” in a tumor suppressor gene express an altered mRNA repertoire that may identify targets for measures that could delay or even prevent progression to carcinoma. We report here on the transcriptomes of primary breast and ovarian epithelial cells cultured from BRCA1 and BRCA2 mutation carriers and controls. Our comparison analyses identified multiple changes in gene expression, in both tissues for both mutations, which were validated independently by real-time reverse transcription-PCR analysis. Several of the differentially expressed genes had been previously proposed as cancer markers, including mammaglobin in breast cancer and serum amyloid in ovarian cancer. These findings show that heterozygosity for a mutant tumor suppressor gene can alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner; these detectable effects of “one hit” represent early molecular changes in tumorigenesis that may serve as novel biomarkers of cancer risk and as targets for chemoprevention. Cancer Prev Res; 3(1); 48–61

One-Hit Effects in Cancer: Altered Proteome of Morphologically Normal Colon Crypts in Familial Adenomatous Polyposis

We studied patients with Familial Adenomatous Polyposis (FAP) because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry, and validation by two-dimensional gel Western blotting. Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription, and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention.

Localization of the interferon-α gene cluster to rat chromosome bands 5q31→q33 by fluorescence in situ hybridization

The chromosomal location of the rat interferon-α (IFNA) gene cluster was determined by fluorescence in situ hybridization. The fluorescent signals were localized to 5q31→q33. A previous report, using

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection

Analysis of cell‐specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols—the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in‐house protocol have been assessed by conducting microarray analyses. Cy3‐labeled total RNA from the colorectal cell line Colo‐205 was compared to Cy5‐labeled Colo‐205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in‐house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross‐sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo‐205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM‐derived material. Taken together, these results demonstrate that LCM‐derived tissue from histological specimens can generate abundant amounts of high‐quality aRNA for subsequent microarray analysis. J. Cell. Physiol. 201: 366–373, 2004.