hela cells 50 years on

8/8/2019 HeLa Cells 50 Years On

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P E R S P E C T I V E S

To cancer research, HeLa is the equivalent of the goose that laid the golden egg aconstant supply of a precious and essential resource. Within a few years, HeLa cells had beendistributed worldwide and became the laboratory model of the cancer cell that would be used for

much of cancer research. But they are not just used for cancer research HeLa cells are usedthroughout biomedical research to study the biochemical pathways of normal and diseased tissuein human cells. Although thousands of continuous cell lines from almost every type of humancancer have since been established mainly in the 1970s and 1980s 9 HeLa is still the most

widely used human cancer cell line. What was special about the cancer from which HeLa cells were grown? Generally, the human cancers that grow permanently in culture are a selected groupof very aggressive cancers that have acquired the necessary phenotypic and genotypic changes 10.

Almost all of the continuous cell lines are derived from high-grade, high-stage cancers. It ispossible to grow some less aggressive cancers permanently, but very few scientists have had thepatience, tenacity or skills to overcome the technical hurdles 11. A short cut is to immortalize thecells with viral genes, the products of which bind and inhibit key proteins such as p53 and

retinoblastoma (RB). So why do normal human cells usually senesce and die, rather than undergospontaneous transformation in vitro to produce permanent cultures, as normal rodent cells sooften do? One possibility is that the difference is related to the higher capacity of human cells forDNA repair 12.

Why was human cancer in a test tube such an important goal? With cell lines, it ispossible to go back to the same cancer again and again, bladder was almost entirely replaced withthe tumour. By this time, her cancer was also growing like wildfire in the laboratory. The cell line

was called HeLa, taken from the first two letters of Henrietta Lacks names.

The failure to preserve complete anonymity was regrettable, but, to give a measure of

confidentiality, the donor was said to be Helen Lane or Helen Larson. It was not customary thento ask for written permission to obtain such samples for research purposes, and there is norecord that Henrietta Lacks consented to the use of her cells. Attitudes were different then prison inmates were shown on television being injected with HeLa cells, proud that they wererepaying some of their debt to society (The Way of All Flesh, BBC TV documentary screened on19 March 1997 in the United Kingdom).

When Mrs Lacks children event ually discovered more than 20 years later what hadhappened to her tissue, they were shocked that cells from their mother had been distributed

worldwide and no one had ever sought their views or permission. The requirement today fordocumented patient consent for research samples is, in part, a consequence of the HeLa cell

story.

The Good

Fifty years ago, the HeLa cell was seen as a great breakthrough, and possibly even the key to a cure for human cancer. The war on cancer and the worldwide hunt for the virus that wasbelieved by some scientists to cause human cancer was soon to follow. Our knowledge of every fundamental process that occurs in human cells whether normal or abnormal hasdepended to a large extent on using HeLa and other cell lines as a model system. Much of what

we know today, and much of what we do tomorrow, depends on the supply of HeLa and othercell lines.

George Gey was a brilliant and highly respected scientist. By 1951, he had been growing cells for nearly 30 years, and until 1937 had worked with Warren Lewis. He was the first to show


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in vitro transformation; he also made some of the first phase-microscope time-lapse films of living cells and developed roller tubes for culturing cells. His goal was to cure cancer, and he didnot take time out to write papers. Crucially for the success of HeLa cells his wife Margaret

was the chief technician and the meticulous director of day -to-day operations in the laboratory.

TIMELINE

1907 -- Ross G. Harrison develops th e hanging drop culture to study frog nerve -cell growth.1910 -- Montrose T. Burrows and Alexis Carrel grow chick embryo cells in tissue culture.1940 -- Wilton R. Earle and George Gey generate a rodent continuous cell line.1951 -- Klaus H. Rothfels and colleagues show interspecies cross-contamination.1958 -- George and Margaret Gey and Mary Kubicek develop HeLa, the first human cancercontinuous cell line.1967 -- Walter Nelson-Rees shows widespread HeLa cross-contamination.1974 -- Cross-contaminated cell lines are used at record levels.

1990 -- Dennis Gilbert, Stephen OBrien and colleagues apply multilocus DNA fingerprinting tocell-line authentication.2002 -- Stan Gartler shows intraspecies cross-contamination.

Timeline | The development of human cancer cell linesFigure 1 | George Gey. Courtesy of Alan Mason

Chesney Medical Archives.

P E R S P E C T I V E S

The Bad

Once George Gey had shown that it was possible to culture human cancers, everyone was able to do it. Suddenly, not only human cancers could be cultured, but also normal humancells became spontaneously transformed and proliferated at great speed in the laboratory. Thenumber of cells could double about every 24 hours, and soon this was happening in biomedicallaboratories worldwide.

But of course it was not that easy to establish a cell line from a human cancer itremains very difficult to this day for most types of cancer and normal human cells almostnever spontaneously transform. It soon became clear that many of the cell lines were not whatthey were claimed to be. Monkey cells turned out to be human cells; human cells were shown tobe mouse cells 18.

But it took more than 15 years before the full extent of the problem was revealed. Until1967, it had not been possible to distinguish between cell lines that were derived from differentindividuals of the same species. Stan Gartler then introduced the concept of biochemicalpolymorphism to the study of human cell lines 19 : some proteins have several different forms,and these forms can differ between individuals.

In 1962, the American Type Culture Collection (ATCC) was set up to collect authenticcell cultures. Stan Gartler was supplied 18 supposedly unique human cell lines by the ATCC andother sources, and investigated the expression of the enzyme glucose-6-phosphatedehydrogenase.


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Individuals have either the A or the B form of the enzyme, and these are distinguishedaccording to mobility in a gel. The A form is almost exclusively found in black individuals, at anincidence of ~30%. HeLa and the other 17 cell lines that were tested expressed the type A form,and also had an identical phenotype for another polymorphic enzyme, phosphoglucomutase 1.Stan Gartler suggested that perhaps all of these

cell lines were HeLa cells 20.

So most of the new human cell lines that had been established since George Geyssuccess back in 1951 were not new, they were just more HeLa cells. Scientists in dozens of laboratories had been careless and mixed up the cells. But Stan Gartler might as well have talkedto himself even scientists who must have known that his conclusions were correct attackedhim. Too many people had written grants and publications on the basis of the false cell linesfrom the ATCC to admit that there might be a problem. and have an endless supply of cells.Genetic drift and pheno-typic change will be minimal within a laboratory 13 , provided that thecells are not grown continuously instead, the cells should be replenished from frozen stocksevery few weeks and standard quality control measures are used.

A chromosomal analysis has show thatthe HeLa genome has been remarkably stableafter years of continuous cultivation 14 . However, it is also relatively easy to select strains of HeLa that have particular properties by applying selection pressures deliberately oraccidentally simply by altering the culture conditions, such as the medium or serum. Forexample, it is possible to select HeLa cells that grow in suspension rather than attached to theculture dish, or HeLa cells that are resistant to cancer drugs.

One of the first applications of HeLa cells was in the fight against polio. GeorgeGey and colleagues in Minneapolis showed that polio virus grew easily in HeLa cells, and killedthe cells, which provided a simple diagnostic test 15 . Large numbers of cells were needed to grow

the virus in order to produce the polio vaccine that Jonas Salk subsequently developed. A HeLaproduction facility was set up at the Tuskegee Institute in Minnesota which was not ideal, asboth the summer and winter temperatures could be lethal to the cells during shipment.

Nevertheless, about 600,000 cultures had been shipped within two years 16 . Jonas Salk even injected some patients with HeLa cells, although at the time he thought that he wasgrowing the vaccine in normal monkey cells 7. HeLa cells are even more importanttoday than when they were first described. Every year for the past 20 years the number of citations for HeLa on MedLine has increased, with more than four times as many hits in the year2000 as in 1980. Many more publications use HeLa cells without acknowledgement (see BOX 1).

HeLa and the other human cancer cell lines that have been established since 1951 are thebedrock of laboratory cancer research. Analysis of the frequency of use of cell lines in papersthat were published in one recent issue of Cancer Research indicated that three-quarters of thepublications used cell lines, and, in total, more than 112 cell lines had been used 17.

Perhaps the main reason underlying the continued use of false cell lines is certain cell-linebanks. Despite being aware of the problem and being the most frequent source of cells, somehave continued to sell cells under false descriptions. The small print has sometimes indicated thatthe false cell line might have HeLa characteristics, which in itself is misleading of courseHeLa cells have HeLa characteristics.

What is the significance of the rising mountain of incorrect data? The implication is that~20% of publications using cell lines contain false data, but that does not mean that all of these


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Box 1 | A sample of the better known HeLa cell cross-contaminants

Comprehensive lists of ~100 early examples of cross-contamination are given in thereferences of Walter Nelson-Rees 21 23. The cell lines described briefly in this box are authenticHeLa cells, and there is no evidence that they contain any genetic information from any other

cell type.

As a result of genetic drift and being subjected to different conditions in variouslaboratories, each strain might have genetic and phenotypic differences. There is no commonstock of HeLa cells, and consequently every batch from each source will be slightly different.

The differences between the various cell stocks labelled HeLa are probably as great as thedifferences between these various strains given different names. Within the last year, some of thefalse cell lines listed below were catalogued and sold by some cell banks under the false nameand false description.

HeLa (KB). The HeLa subline KB was thought to be derived from an oral cancer 33 . It was

cited more than 300 times during the period 1998 2000 in MedLine, and some of these studiesused the cells as a model of skin or head and neck cancer 34,35. Few of the papers mention orseem to be aware that the cells are derived from a glandular cancer of the cervix.

HeLa (HEp -2). The HeLa subline HEp-2 was thought to be derived from a cancer of the larynx36. It was cited more than 300 times during the period 1998 2000 in MedLine and is frequently used by virologists as a human epithelial cell line37,38. Usually, there is no mention that these cellsare a HeLa subline and are derived from a cervical cancer.

HeLa (WISH), HeLa (AV 3) HeLa (FL). These three sublines of HeLa were all thought to bederived from amnion cells, the most well-known and widely used one being WISH 39. Despite

their origin from cervical cancer, these cell lines are sometimes used in the fields of reproductionand endocrinology, and are described as being normal human amnion cells 40,41.

HeLa (L132). The HeLa subline L132 was thought to be derived from normal humanembryonic lung cells 42. These HeLa cells are sometimes described as being normal embryonichuman lung epithelial cells 43,44.

HeLa (Intestine 407). The HeLa subline INT 407 was thought to be derived from humanintestinal epithelial cells45 . Despite its origin from a cancer of the cervix, it is still used as amodel of normal human gastrointestinal cells 46,47.

HeLa (Chang liver). The HeLa subline called Chang liver was thought to be derived fromnormal liver cells 48. Despite being cervical cancer cells, Chang liver cells are sometimes used instudies of hepatic-cell physiology 49,50.

Whether or not it was HeLa or another cell line does not seem important. However, inthose cases in which the investigator has assumed a specific tissue origin of the cell line (such asliver or lymphocytes), the work is of dubious value 20. There is, at present, a campaign to havethe false cell lines renamed with their correct designation 32.

The future


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HeLa cells are even more important today than they were when first grown by GeorgeGey 50 years ago. Cell lines have been, and will continue to be, the model system of the cancercell that is used by most cancer research scientists. However, the HeLa story also shows theconsequences when peer review fails and there is a lack of quality control.

John R. Masters is at the Institute of Urology,University College London,67 Riding House Street,London W1W 7EY, UK.

e-mail: [email protected] DOI: 10.1038/nrc775

1. Lewis, W. H. Malignant cells. The Harvey Lectures 31, 214 234 (1936).2. Harrison, R. G. Observations on the living developing nerve fiber. Proc. Soc. Exp. Biol. Med.4, 140 143 (1907).

3. Carrel, A. On the permanent life of tissues outside the organism. J. Exp. Med. 15, 516 528(1912).4. Burrows, M. T. The cultivation of tissues of the chick embryo outside the body. J. Am. Med.

Assoc. 55, 2057 2058 (1910).5. Earle, W. R. et al. Production of malignancy in vitro. IV. The mouse fibroblast cultures andchanges seen in living cells. J. Natl Cancer Inst. 4, 165 212 (1943).6. Gey, G. O., Coffman, W. D. & Kubicek, M. T. Tissue culture studies of the proliferativecapacity of cervical carcinoma and normal epithelium. Cancer Res. 12, 264 265 (1952).7. Gold, M. A Conspiracy of Cells. One Womans Immortal Legacy and the Scandal it Caused(State Univ. New York Press, New York, 1986).8. Jones, H. W., McKusick, V. A., Harper, P. S. & Wuu K. D. The HeLa cell and a reappraisal of

its origin. Obstet. Gynecol. 38, 945 949 (1971).9. Masters, J. R. W. & Palsson, B. . (eds) Human Cell Culture Vol. 1 3 (Kluwer Academic,Dordrecht, 1999).10. Masters, J. R. W. Human cancer cell lines: fact and fantasy. Nature Rev. Mol. Cell Biol. 1,233 236 (2000).11. Wistuba, I. I. et al. Comparison of features of human breast cancer cell lines and theircorresponding tumours. Clin. Cancer Res. 4, 2931 2938 (1998).12. Sanford, K. K. & Evans, V. J. A quest for the mechanism of spontaneous malignanttransformation in culture with associated advances in culture technology. J. Natl Cancer Inst. 68,895 913 (1982).13. UKCCCR guidelines for the use of cancer cell lines in cancer research. Br. J. Cancer 82,

1495 1509 (2000).14. Macville, M. et al. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res. 59, 141 150 (1999).15. Scherer, W. F., Syverton, J. T. & Gey, G. O. Studies on the propagation in vitro of poliomyelitis viruses. J. Exp. Med.97, 695 709 (1953).16. Brown, R. W. & Henderson, J. H. M. The mass production and distribution of HeLa cells at

Tuskegee Institute, 1953 55. J. Hist. Med. Allied Sci. 38, 415 431(1983).17. Arlett, C. F. The use of dubious cell lines in research: is trust enough? Lancet Oncol. 2, 467(2001).18. Rothfels, K. H., Axelrad, A. A., Siminovitch, L., McCulloch, E. A. & Parker, R. C. in Proc.3rd Canadian Cancer Research Conference 1958 (ed. Begg, R. W.) 189 214 (Academic, New

York, 1958).19. Gartler, S. M. Genetic markers as tracers in cell culture. Natl Cancer Inst. Monogr. 26, 167


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embryo. Fed. Proc. 19, 386 (1960).43. Kasper, M. et al. Induction of apoptosis by glyoxal in human embryonic lung epithelial cellline L132. Am. J. Respir. Cell Mol. Biol. 23, 485 491 (2000).20. Gartler, S. M. Apparent HeLa cell contamination of human heteroploid cell lines. Nature217, 750 751 (1968).

21. Nelson-Rees, W. A., Flandermeyer, R. R. & Hawthorne, P. K. Banded marker chromosomesas indicators of intraspecies cellular contamination. Science 184, 1093 (1974).22. Nelson-Rees, W. A. & Flandermeyer, R. R. HeLa cultures defined. Science 191, 96 98(1976).23. Nelson-Rees, W. A., Daniels, D. W. & Flandermeyer, R. R. Cross-contamination of cells inculture. Science 212, 446 452 (1981).24. Nelson-Rees, W. A. Responsibility for truth in research. Phil. Trans. R. Soc. Lond. B 356,849 851 (2001).25. Editorial. Responsibility for trust in research. Nature 289, 211 212 (1981).26. Markovic, O. & Markovic, N. Cell cross-contamination in cell cultures: the silent andneglected danger. In Vitro Cell Dev. Biol. 34, 1 8 (1998).

27. MacLeod, R. A. F. et al. Widespread intraspecies cross-contamination of human tumour celllines. Int. J. Cancer 83, 555 563 (1999).28. Dirks, W. G., MacLeod, R. A. & Drexler, H. G. ECV304 (endothelial) is really T24 (bladdercarcinoma): cell line cross-contamination at source. In Vitro Cell Dev. Biol. 35, 558 559 (1999).29. Drexler, H. G., Uphoff, C. C., Dirks, W. G. & MacLeod, R. A. F. Mix ups and mycoplasma:the enemies within. Leukemia Res. (in the press).30. Drexler, H. G., MacLeod, R. A. F. & Dirks, W. G. Cross-contamination: HS-Sultan is not amyeloma but a Burkitt lymphoma cell line. Blood 98, 3495 3496 (2001).31. Masters, J. R. W. et al. STR profiling provides an international reference standard for humancell lines. Proc. Natl Acad. Sci. USA 98, 8012 8017 (2001).32. Stacey, G. N. et al. Cell contamination leads to inaccurate data: we must take action now.

Nature 403, 356 (2000).

hela cells 50 years on

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