The HeLa Bomb
As Stanley Gartler stepped up to the microphone in the meeting room of the palatial Bedford Springs Hotel, one of the final remaining great nineteenth century spa hotels in the United States, he would have been forgiven for feeling a little nervous. He is speaking at the Second Decennial Review Conference on Cell Tissue and Organ Culture in September 1966. Geneticists, cell culture researchers and giants from rapidly growing biotechnology companies have congregated to discuss the field of cell culture and its potential medical applications, which ranged from curing cancer, to creating clones and hybrid creatures. Stanley Gartler, however, does not share the optimism spreading around the room. He is about to give a presentation that could send this burgeoning field crashing down around its very founders. He is about to drop ‘the HeLa bomb’.
15 years earlier, the ability to grow human cells in the lab for further study was an impossibility. Many had tried, but human cells simply died the moment they were taken out of the body. The need for a stable human cell line was clear; with a plentiful supply of cells that could grow and divide in laboratory conditions, scientists would be able to study the processes that underlay diseases such as cancer and devise and test therapies and prophylactics without the need to experiment on living subjects. The field of cell culture got its first big break in 1951, when a woman called Henrietta Lacks walked through the doors of Johns Hopkins Hospital in Baltimore. Henrietta had been complaining of a “knot” in her stomach1 but had been told by her friends not to worry, that the tightness and cramping she felt was due to pregnancy. While Henrietta was indeed pregnant, the “knot” only got worse once she gave birth, during which she experienced a massive haemorrhage, prompting her physician to refer her to Johns Hopkins. It was here that a
node was discovered on her cervix and she was diagnosed with a cervical carcinoma. During her treatment, which involved the insertion of radium tubes into her vagina and heavy doses of X-rays, samples of both healthy and cancerous cervical tissue were taken without her knowledge or consent. These cells were handed over to George Gey, a cancer researcher at the hospital, who was attempting to grow the first ever ‘immortal’ human cell line. Henrietta’s cells were the lucky break he had been waiting for. They proved to be highly aggressive, able to grow in simple culture mediums and, crucially, able to divide over and over again, without end. Gey had produced the first ever immortal human cell line, which opened the doors for a rapid growth in the field of cell culture. He named his cell line HeLa, and it remains one of the most commonly studied cell lines in biomedical science to this day. Henrietta died in late 1951, her body scarred by aggressive, rudimentary cancer treatment and her insides dotted with black pearls of cancer that had spread throughout every major organ. She and her family had no idea of the contribution Henrietta had made to modern science, nor did they receive a penny of compensation from the companies that were set up to sell HeLa cells to a desperate audience of researchers.
George Gey was in the audience as Stanley Gartler stepped up to speak. As were the numerous other geneticists that had gone on to create their own cell lines, including giants of the field Robert Shihman Chang and Leonard Hayflick, whose WISH cell line had originated from the amniotic sac of his unborn daughter2. They were all shocked when Gartler dropped his ‘HeLa bomb’, in which he stated that he believed that 18 of the most commonly used cell lines were all contaminated with one type of cell – HeLa. He posited that since all the cell lines that he tested returned a positive for a genetic marker called Glucose-6-Phosphate Dehydrogenase A (G6PD-A) that was present in a small proportion of black Americans, HeLa must have contaminated and taken over all the cell lines that had been at the centre of medical research for over a decade3. The shock in the room was palpable, with numerous researchers firing questions at Gartler, desperately attempting to prove him wrong. The consequences were substantial: researchers who thought that they had been exploring the underlying causes of specific cancers of the liver, lungs, skin and more and determining the effect of different therapies on cell type-specific cancers had in fact been working on the same cells as each other. The potentially wasted hours, resources and dollars didn’t bear thinking about.
A Contamination Case Study: Chang Liver
Dr Robert Shihman Chang was born in Quangdong, China in 1922. He came to the United States just after the cessation of World War 2 and went on to be one of the pioneers of cell culture and a major player in the search for a viral cause for cancer. Dr Chang pioneered the use of Epstein-Barr virus to immortalise lymphoid cells (cells of the immune system and their naïve precursors) – mimicking a process that occurs in some blood cancers, such as Hodgkin’s Lymphoma and Burkitt’s Lymphoma.
He was among of the first cell culturists to produce his own cell line after George Gey had successfully produced HeLa. Called Chang Liver, this cell line was intended to be a source of immortal healthy liver cells. Chang Liver was one of the cell lines that Stanley Gartler claimed to be contaminated in his infamous ‘HeLa bomb’ speech, as Chang’s cell line displayed the rare G6PD-A tag (among others) that were present in HeLa. Chang’s reputation – and that of his cell line – was further damaged when Walter Nelson-Rees placed him on his second ‘hit-list’, as his cells contained distinctive chromosome sections unique to those found in HeLa cells.
Chang, however, argued fervently for the authenticity of his cells. His laboratory, which Stanley Gartler noted contained a large volume of HeLa cells, highlighted that Chang Liver cells were producing enzymes normally produced only by liver cells. In doing so, Chang had highlighted a function of cancer cells called ‘dedifferentiation’, in which cancerous cells lose their specialised function and enter a more plastic, stem cell-like state. Depending on the conditions they are subjected to, such as being treated as if they were liver cells, these ‘dedifferentiated’ cells can then go on to gain functions, such as enzyme production, that the original, healthy cells from which they are derived could not carry out.
The final death knell for Chang Liver was the discovery of a series of identical short tandem repeats in both HeLa cells and Chang’s cell line by Masters et al. in 2001. Today, Chang Liver is one of the 400 cell lines marked by the ICLAC as misidentified or contaminated. However, it still continues to be used today without any mention of HeLa contamination by the vast majority of researchers.
Further case studies of misidentified cell lines can be found on the ICLAC website.
The Hit List
It was a major setback that was highlighted further just under decade later by Walter Nelson-Rees et al. in their cell culture contamination exposé of 19744, in which they reported substantial contamination of further commonly used cell lines by HeLa. In these early years of cell culture, scientists were aware of potential bacterial and viral contamination and even that of cells other species, but nobody had ever thought to test for contamination by other cells of human origin. Many researchers had been working on generating their own cell lines in labs that also had large volumes of HeLa stored in them, as most molecular biology labs did at the time. HeLa cells are so hardy and invasive that they can survive on unwashed hands, labcoats and shoes and a single cell is enough to contaminate an entire culture. The work of Gartler and Nelson-Rees should have brought about a shift in cell culture and biomedical research standards, but it didn’t. Indeed, Nelson-Rees left scientific research in 1981 partly due to increasing pressure from other scientists, furious that their both their cell lines and names had made his regular “hit-lists”5. Indeed, Michael Gold claims in his book ‘A Conspiracy of Cells’ that:
Nelson-Rees was weary of the battle. He was in a business that generated enemies, not allies.6
What should have been a watershed moment in the history of biomedical research was instead met with resistance and outrage. Surely, though, with modern DNA fingerprinting techniques and with an inevitable refinement of cell culture practices having taken place, this is a problem of the past? The answer to that according to a new paper published this month in PLOS One, says the answer to that question is a resounding no. In fact, the problem is getting worse.
The Infected Literature
According to a new study published in October 2017, over 32,000 published articles either directly or indirectly report on research that used misidentified or contaminated cell lines. Authors Serge Horbach and Willem Halfmann highlight that there are major problems that arise when using a misidentified cell line, including “errors, false conclusions and irreproducible results” 7. Horbach and Halfmann were concerned about not only the number of primary research papers using contaminated cell cultures, but also about the number of secondary articles referring to this work.
Having undertaken an exhaustive search using the Web of Science database, they discovered that 92% of articles containing tainted cell lines are cited by another paper, with total citations amassing 500,000.
Horbach and Halfmann warn that the impact could be substantial. Most ‘tainted’ articles appear in the fields of oncology, biochemistry, cell biology and molecular pathology, highlighting serious questions about medical and pharmaceutical developments that could have been made in response. Almost more concerning are the tendrils of contamination infecting secondary literature, contributing to the spread of false data. Horbach and Halfmann explain that ‘tainted’ secondary literature can be found in fields as diverse as agricultural science and engineering, where authors may be less aware of the origin of the cell lines used in the research that they are citing.
It is important to note that a vast majority of the researchers using tainted cell lines are not doing so with the aim of deceiving their audiences. In all likelihood, the majority of the scientists will have made honest mistakes, overlooking the need to check the status of the cell lines they are using. Additionally, some research does not require the use of specific cell lines from a certain type of cell. If, for example, researchers are interested in a ubiquitous cellular process the origin of the cell line they are using has little relevance.
Horbach and Halfmann warn that the scale of the problem that they have highlighted may well have been underestimated7. Their method of counting does not include tainted cell lines that we do not yet know about and only used one spelling for each cell line – papers that used a different spelling to those in the ICLAC (International Cell Line Authentication Committee) database would have been missed.
But what actions can be taken to address this growing problem? Clearly, catching the contamination upstream will be the key to cleansing the literature. Researchers must be encouraged to actively check the status of the cell lines they are using and clearly indicate this in their paper. In recent years it has also become far easier to confirm the identity of the cells being used
with the discovery and refinement of short tandem repeat (STR) analysis (see Figure 3). There is an argument to withdraw all the tainted papers from circulation in the wider literature, but, as Walter Nelson-Rees discovered, the negative impact this may have on the careers of researchers who may have made an honest mistake may lead to further resistance from the scientific community. A fairer solution, Horbach and Halfmann argue, would be to simply place a note against all of the identified papers indicating that the work was carried out using a misidentified cell line.
Many watershed moments in the history of cell culture have come and gone without action from the wider scientific community. From Stanley Gartler’s ‘HeLa bomb’ in 1966, to Walter Nelson-Rees’ hit-lists of the 1970s and 1980s and finally the invention of advanced technologies that allow for the accurate identification of cell lines, there have been a myriad of opportunities to end the contamination of scientific literature by misidentified cell lines. With this latest bombshell, the time has surely come to take action to protect the integrity of the crucial biomedical research taking place around the world.
‘Til next time…
1. Skloot R. The Immortal Life of Henrietta Lacks. First ed. New York: Crown Publishing Group; 2010. 381 ch.10
2. Bioinformatics SSIo. WISH (RRID:CVCL_1909) 2015 [Available from: http://web.expasy.org/cellosaurus/CVCL_1909.
3. Gartler SM. Apparent HeLa Cell Contamination of Human Heteroploid Cell Lines. Nature. 1968;217(5130):750-1.
4. Nelson-Rees WA, Flandermeyer RR, Hawthorne PK. Banded Marker Chromosomes as Indicators of Intraspecies Cellular Contamination. Science. 1974;184(4141):1093.
5. Gold M. A Conspiracy of Cells. Albany, New York: State University of New York Press; 1986. p. 100-1.
6. Gold M. A Conspiracy of Cells. Albany, New York: State University of New York Press; 1986. p. 142-3.
7. Horbach SPJM, Halffman W. The ghosts of HeLa: How cell line misidentification contaminates the scientific literature. PLOS ONE. 2017;12(10):e0186281.