In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.

/pdf/normal-keratinization-in-a-spontaneously-immortalized-5753ggq1uq.pdf

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.

NOTE The report of the Committee without its annexes appears as Official Records of the General Assembly, Sixty-third Session, Supplement No. 46. The designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the United Nations concerning the legal status of any country, territory, city or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries. The country names used in this document are, in most cases, those that were in use at the time the data were collected or the text prepared. In other cases, however, the names have been updated, where this was possible and appropriate, to reflect political changes. Scientific Annexes Annex A. Medical radiation exposures Annex B. Exposures of the public and workers from various sources of radiation INTROdUCTION 1. In the course of the research and development for and the application of atomic energy and nuclear technologies, a number of radiation accidents have occurred. Some of these accidents have resulted in significant health effects and occasionally in fatal outcomes. The application of technologies that make use of radiation is increasingly widespread around the world. Millions of people have occupations related to the use of radiation, and hundreds of millions of individuals benefit from these uses. Facilities using intense radiation sources for energy production and for purposes such as radiotherapy, sterilization of products, preservation of foodstuffs and gamma radiography require special care in the design and operation of equipment to avoid radiation injury to workers or to the public. Experience has shown that such technology is generally used safely, but on occasion controls have been circumvented and serious radiation accidents have ensued. 2. Reviews of radiation exposures from accidents have been presented in previous UNSCEAR reports. The last report containing an exclusive chapter on exposures from accidents was the UNSCEAR 1993 Report [U6]. 3. This annex is aimed at providing a sound basis for conclusions regarding the number of significant radiation accidents that have occurred, the corresponding levels of radiation exposures and numbers of deaths and injuries, and the general trends for various practices. Its conclusions are to be seen in the context of the Committee's overall evaluations of the levels and effects of exposure to ionizing radiation. 4. The Committee's evaluations of public, occupational and medical diagnostic exposures are mostly concerned with chronic exposures of …

sources and effects of ionizing radiation

http://eldeirylab.com/resources/reprints/1990-1994/Kastan_Cell_1992.pdf

A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia

The human diet contains a great variety of natural mutagens and carcinogens, as well as many natural antimutagens and anticarcinogens. Many of these mutagens and carcinogens may act through the generation of oxygen radicals. Oxygen radicals may also play a major role as endogenous initiators of degenerative processes, such as DNA damage and mutation (and promotion), that may be related to cancer, heart disease, and aging. Dietary intake of natural antioxidants could be an important aspect of the body’s defense mechanism against these agents. Many antioxidants are being identified as anticarcinogens. Characterizing and optimizing such defense systems may be an important part of a strategy of minimizing cancer and other age-related diseases.

https://science.sciencemag.org/content/sci/221/4617/1256.full.pdf

Dietary carcinogens and anticarcinogens Oxygen radicals and degenerative diseases

Multiple genetic changes occur during the evolution of normal cells into cancer cells. This evolution is facilitated in cancer cells by loss of fidelity in the processes that replicate, repair, and segregate the genome. Recent advances in our understanding of the cell cycle reveal how fidelity is normally achieved by the coordinated activity of cyclin-dependent kinases, checkpoint controls, and repair pathways and how this fidelity can be abrogated by specific genetic changes. These insights suggest molecular mechanisms for cellular transformation and may help to identify potential targets for improved cancer therapies.

https://science.sciencemag.org/content/sci/266/5192/1821.full.pdf

Cell cycle control and cancer

The induction of sister chromatid exchanges (SCE) was examined in Chinese hamster ovary cells irradiated in the G1 phase of the cell cycle with alpha-particles from a plutonium-238 source. A significant increase in the frequency of SCE occurred with doses as low as 0.31 mGy (31 millirads). Although 30% of the cells showed an increased frequency of SCE at this dose, less than 1% of cell nuclei were actually traversed by an alpha-particle. A dose of approximately 2.0 Gy was necessary to produce a similar increase in SCE by X-rays. These results indicate that genetic damage may be induced by low doses of alpha-radiation in cell nuclei not actually traversed by an alpha-particle. This phenomenon may have important implications in the estimation of risks of such exposures.

https://cancerres.aacrjournals.org/content/canres/52/22/6394.full.pdf

Induction of sister chromatid exchanges by extremely low doses of alpha-particles.

We examined the induction of HPRT mutations in CHO cells exposed to low fluences of (238)Pu alpha particles from a specially constructed irradiator. The dose-response relationship was linear over the dose range of 5 cGy-1.2 Gy. However, unexpected sensitivity, leading to a significantly higher frequency of mutations than would be predicted by a back extrapolation from the data for higher doses, was observed in the dose range below 5 cGy, where the mean number of alpha-particle traversals per nucleus was significantly less than one (0.05-0.3). The frequency of mutations induced by a single alpha particle traversing the nucleus of a cell was increased nearly fivefold at the lowest fluence studied. The data are consistent with the conclusion that the enhanced efficiency of each nuclear traversal at low particle fluences is the result of mutations arising in nonirradiated, bystander cells.

https://www.jstor.org/stable/pdfplus/3580153.pdf

Unexpected sensitivity to the induction of mutations by very low doses of alpha-particle radiation: evidence for a bystander effect.

Ku70: A Candidate Tumor Suppressor Gene for Murine T Cell Lymphoma

The frequency of mutations at the Hprt locus was measured in clonal populations of Chinese hamster ovary cells derived from single cells surviving exposure to 0-12 Gy of X rays or 2 Gy of alpha particles. Approximately 8-9% of 446 clonal populations examined 23 population doublings after irradiation showed high frequencies of late-arising mutations as indicated by mutant fractions 10(2)-10(4)-fold above background. The frequency with which such clones occurred was similar for alpha-particle irradiation and X irradiation, with no apparent dose dependence for X irradiation over the range of 4-12 Gy. The molecular structure of Hprt mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. Of mutations induced directly after exposure to X rays, 75% involved partial or total gene deletions. Only 19-23% of late-arising (delayed) mutations were associated with deletions, the preponderance of these being partial deletions involving one or two exons. This spectrum was very similar to that for spontaneously arising mutations. To determine whether delayed mutations were non-clonal, the spectrum of exons deleted was examined among 29 mutants with partial deletions derived from a single clonal population. The results indicated that at least 15 of these mutants arose independently. To examine the relationship between the occurrence of delayed mutations and chromosomal instability, 60 Hprt mutant subclones isolated from a clonal population showing a high frequency of delayed mutations were serially cultivated in vitro. Of these, 14 showed a slow-growth phenotype with a high frequency of polyploid cells (10-38%) and a markedly enhanced frequency of non-clonal chromosomal rearrangements including both chromosome-type and chromatid-type aberrations. These clones also showed a 3- to 30-fold increase in the frequency of ouabain-resistant mutations; no ouabain-resistant mutants were induced directly by X irradiation. These results suggest that among clones showing a high frequency of delayed mutations there may be a subpopulation of cells that are particularly unstable; selection for the slow-growth phenotype has the effect of selecting for this chromosomally unstable subpopulation.

Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of X rays and alpha particles.

SummarySynchronous Chinese hamster cells exposed to heat treatments of 45·5°c were very sensitive during S phase when chromosomal damage could account for cell killing, i.e. as for x-irradiated cells, the 37 per cent survival value for heated cells was observed when there was about one aberration per cell. However, the aberration frequency was very low and could not account for cell killing when the cells were heated, either during mitosis when they were most sensitive in terms of lethality, or during G1 when they were most resistant. Furthermore, about 50 per cent of the deletions in these cells heated in mitosis or G, occurred at the secondary constriction of the X chromosome. Incorporation of BUdR into DNA prior to synchronizing the cells had only an additive interaction with heat administered during G1 or S. This interaction was characterized by a reduction in the shoulder of the dose-response curves observed for both chromosomal aberrations and cell killing.These findings contrast greatly with those ...

Hatsumi Nagasawa

Papers

Induction of sister chromatid exchanges by extremely low doses of alpha-particles.

Unexpected sensitivity to the induction of mutations by very low doses of alpha-particle radiation: evidence for a bystander effect.

Ku70: A Candidate Tumor Suppressor Gene for Murine T Cell Lymphoma

Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of X rays and alpha particles.

Heat-induced lethality and chromosomal damage in synchronized Chinese hamster cells treated with 5-bromodeoxyuridine.