HeLa RNA is everywhere

Immortal LifeThe first immortal human cell line ever produced, HeLa, originated from a cervical adenocarcinoma taken from Henrietta Lacks. The cell line grew so well that it was used in many laboratories and soon was found to contaminate other cell lines. Now HeLa RNA has made its way into human sequence databases.

Although the cause of Henrietta Lacks’ cervical tumor was not known in her lifetime, we now understand that it was triggered by infection with human papillomavirus (HPV) type 18. When this virus infects the cervical epithelium, the viral DNA may integrate into the host genome, causing the cells to become transformed and eventually malignant. HeLa cells are known to contain integrated HPV18 DNA.

There are many different types of cancer, each caused by errors in DNA. The Cancer Genome Atlas (TCGA) is a database for collecting the DNA sequences of diverse cancers from many different individuals. It was established to help understand what mutations cause various types of cancer. As viruses are known to be responsible for about 20% of human cancers, searching this database for viral sequences can advance our understanding of their role in this disease. For example, almost every genome from patients with cervical cancer contains HPV DNA.

A recent search of the TCGA for viral sequences revealed that, in addition to cervical cancer, HPV18 sequences were found in many other cancers, including colon, head and neck, kidney, liver, lung, ovary, rectum, and stomach. The HPV18 sequences in non-cervical cancers resembled the viral sequence found in HeLa cells, both in integration site and single nucleotide variations. In other words, the HPV18 in these cancers closely matches that of the viral genome integrated into HeLa cells, and their presence is likely due to contamination.

Further analysis revealed that the contaminated samples originated from only two genome sequencing centers, the University of North Carolina Lineberger Comprehensive Cancer Center, and the Michael Smith Genome Sciences Centre of the British Columbia Cancer Agency. All the contamination took place in 2011 and 2012, and was limited to 18 (6%) of the sequencing machines.

The contamination with HeLa nucleic acid was observed only in datasets derived from sequencing of RNA, not DNA. I asked the senior author Jim Pipas how he thought this contamination might have taken place:

I can think of two possibilities. One is that the RNA isolated from the tumor was somehow contaminated with HeLa sequences. The other is that HeLa cell RNA was sequenced on the same machine as the tumors and the contamination is from the sequencing machine itself.

It is well known that nucleic acids can become contaminated during their manipulation in the laboratory. The use of sensitive techniques such as PCR and deep sequencing reveal such contamination when it previously went unnoticed. High profile examples of nucleic acid contamination include the retrovirus XMRV associated with chronic fatigue syndrome, and a virus believed to cause hepatitis (a contaminant from laboratory plasticware).

As virus discoverer Eric Delwart noted on TWiV 86, ‘DNA is a real problem. It’s everywhere’. Apparently so is HeLa cell RNA.

A saga of HeLa cells

HeLa spinnerWe have been using HeLa cells in my laboratory since 1982, when I arrived at Columbia University Medical Center fresh from postdoctoral work with David Baltimore at MIT. I brought with me a line of HeLa cells and used them for 30 years for our research on viruses. Here is a story of how we lost the cells and then found them ten months later.

As everyone knows, the continuous HeLa cell line was derived from a cervical tumor taken from Henrietta Lacks in 1951 (if you don’t know the story, you should read Rebecca Skloot’s The Immortal Life of Henrietta Lacks, or my shorter summary). When I arrived at the Baltimore lab in 1979, they were using cells derived from the S3 clone of HeLa cells that had been produced by Philip Marcus in the 1950s. I write ‘derived from’ because someone at MIT had further  cloned the S3 line and selected one that was particularly susceptible and permissive to poliovirus infection. This was the cell line that I took with me to Columbia in 1982.

Because we use so many HeLa cells each week, we grow them in spinner cultures (pictured). The cells are suspended in a glass bottle in nutrient medium and continuously stirred by a magnetic bar. The spinner bottle is placed on top of a stir plate, which contains a motor that drives a rotating magnet that in turn spins the bar in the bottle. When we need to produce monolayers of cells for experiments, we remove cells from suspension and plate them on plastic dishes. The HeLa S3 clone that we use grows very well in suspension and also forms excellent monolayers on plastic dishes.

Over the years we used the HeLa S3 subclone to conduct experiments with poliovirus, echoviruses, Coxsackieviruses, enteroviruses, rhinoviruses, and encephalomyocarditis virus. The cells could be infected with all these viruses, develop cytopathic effects, and form plaques, allowing titration of virus titers. They have been an essential part of my laboratory. The Wall of Polio is just one example of how important these cells have been for our work.

In December 2012, the spinner went down. The drive belt that turns the magnet in the spinner platform broke overnight; the cells settled out and died. Normally we would simply go to our stock of cells frozen in liquid nitrogen, thaw them out, and be up and working again within a week. Unfortunately, our liquid nitrogen tank had run dry one week in the summer of 2012, and all the cells had died. We tried recovering some of the HeLa cells that were frozen, but what grew out were not the same as our S3 subclone.

In the course of the next 9 months we tried HeLa cells from many different sources – laboratories here at Columbia, the American Type Culture Collection, and our colleagues elsewhere. None of the HeLa cells performed like our S3 subclone. Some HeLa lines did not grow well in spinner; others did, but formed poor monolayers. Still others did not support replication or plaque formation when infected with viruses we work on now – poliovirus, rhinoviruses, and encephalomyocarditis virus. I located former members of the Baltimore laboratory, hoping that they had taken the special HeLa cells with them, but I came up empty handed.

A few weeks ago I received an email from a former student who had heard my pleas for HeLa cell help on This Week in Virology. She remembered bringing some of our HeLa cells to her postdoctoral laboratory in Canada, and freezing them down when she left. I contacted the laboratory and found to my delight that our HeLa cells were indeed frozen there; a kind member of the laboratory grew up a stock of the cells, froze them, and shipped them off to us. I received them a week ago and put them into culture. They were the HeLa cells that I used to know: I recognized their morphology immediately. They grew beautifully as monolayers, and just today I set up a spinner culture. We are all looking forward to using the cells in our virology experiments.

Meanwhile, I have a large stock of belts for the magnetic spinner plate (I had to buy seventeen of them, to meet the $50 minimum order); I have placed 6 vials of the cells in our liquid nitrogen tank; and I plan to freeze additional vials in my colleagues’ freezers in case ours goes down again.

There are several lessons to be learned from this saga. First, because virologists are completely dependent on cells, they must take care that they have a reliable stock. This means having someone in the lab checking the level of liquid nitrogen every day, and ordering a new tank when the level is low. Second, it’s important to keep stocks of cells frozen elsewhere. We were very lucky to find them in Canada. Third, HeLa cell lines are very different. Finally, HeLa cells are special. I don’t know of any other cell line that grows well in spinner, makes beautiful monolayers, and allows us to work with so many different viruses. Thank you, Henrietta Lacks.

TWiV 246: Pandora, pandemics, and privacy

On episode #246 of the science show This Week in Virology, Vincent, Alan, Rich, and Kathy discuss the huge Pandoravirus, virologists planning H7N9 gain of function experiments, and limited access to the HeLa cell genome sequence.

You can find TWiV #246 at www.microbe.tv/twiv.

We recorded this episode of TWiV as a Google hangout on air. Consequently the audio is not the same quality as you might be used to. But the tradeoff is that you can see each of us on video.

 

TWiV 227: Lacks security and bad poultry

On episode #227 of the science show This Week in Virology, the complete TWiV team reviews the controversial publication of the HeLa cell genome, a missing vial of Guanarito virus in a BSL-4 facility, and human infections with avian influenza H7N9 virus.

You can find TWiV #227 at www.microbe.tv/twiv.

Henrietta Lacks (HeLa) genome sequence published then withdrawn

HeLa cellsEarlier this month the European Molecular Biology Laboratory (EMBL) published the DNA sequence of the genome of HeLa cells, the cell line that is widely used for research in virology, cell biology, and many other areas. This cell line was produced from a tumor taken from Henrietta Lacks in 1951. Unfortunately the EMBL did not receive permission from Ms. Lacks’ family to publish her genome sequence, and have withdrawn the information from public databases.

The history of HeLa cells has been well chronicled in Johns Hopkins Magazine and by Rebecca Skloot in The Immortal Life of Henrietta Lacks. In early 1951, Ms. Lacks was found to have a malignant tumor of the cervix. During her examination at Johns Hopkins Hospital in Baltimore, MD, a sample of the tumor was removed and used to produce the HeLa cell line. But Ms. Lacks’ family never learned about the important cells that were derived from her until 24 years after her death.

It is quite clear that permission to publish the HeLa cell genome sequence should have been obtained from the Lacks family. This issue are discussed in an opinion piece by Rebecca Skloot in the New York Times.

I was honored to work with Rebecca Skloot during the preparation of Immortal Life, and I am flattered that Ms. Skloot thanked me in the afterward of the book. I have also written about my work with HeLa cells (that’s me in the photo with a spinner of the cells). You might also be interested in my conversation with Philip Marcus, who was the first to produce single cell clones of HeLa cells.

The Immortal Life of Henrietta Lacks

immortal_lifeShortly after I wrote about my years of experience with HeLa cells, I was contacted by author Rebecca Skloot. One of her many questions was how I knew that I had produced 800 billion HeLa cells in my laboratory over 26 years. I learned that she was writing a book about Henrietta Lacks, whose tumor was the source of HeLa cells in 1951. Subsequently I had the privilege of reading an early draft of her book, The Immortal Life of Henrietta Lacks, which will be published next month.

I thought I knew enough about HeLa cells and their origins, but Rebecca’s book shattered that impression. I’ve worked with the cells all my career and have always appreciated them, and the fact that Henrietta gave science something fabulous, but the back story I didn’t appreciate. How the whole affair deeply affected that family, and what they went through. I want to thank Rebecca for working so hard to get the whole story. And for being nice enough that the family trusted her! She not only vividly portrays what the family went through, but shows what HeLa has meant to science, how unscrupulous people always want to take advantage of others, and the good and bad about science. In the end, I keep coming back to the same question: if we had informed consent laws back then, would Henrietta have said no? If so, it would have been a tremendous loss for science and medicine. Or should I say setback – because eventually there would have been others. That’s how science is: someone always makes the discovery, sooner or later.

There will be a public launch of the book on 1 February at 7pm at McNally Jackson Bookstore in New York City. Rebecca will read a bit from the book, talk about it, sign it, and answer questions. Below are the details of the public event. If you are in the New York area, and have an interest in science, I encourage you to attend. I will certainly be there!

Public Launch Event: Rebecca Skloot Discusses Her New Book “The Immortal Life of Henrietta Lacks”

Award winning science writer Rebecca Skloot discusses and signs her new book, The Immortal Life of Henrietta Lacks. Books available for sale at this launch event one day before the book’s official publication date. Free & open to the public.

Book description: Her name was Henrietta Lacks, but scientists know her as HeLa. She was a poor Southern tobacco farmer who worked the same land as her slave ancestors, yet her cells — taken without her knowledge — became one of the most important tools in medicine. The first immortal human cells grown in culture, they are still alive today, though she has been dead for more than sixty years. If you could pile all HeLa cells ever grown onto a scale, they’d weigh more than 50 million metric tons — more than 100 Empire State Buildings. HeLa cells were vital for developing the polio vaccine; uncovered secrets of cancer, viruses, & the effects of the atom bomb; helped lead to important advances like in vitro fertilization, cloning, and gene mapping; and have been bought and sold by the billions. Yet Henrietta Lacks remains virtually unknown, buried in an unmarked grave. Now Rebecca Skloot takes us on an extraordinary journey, from the colored ward of Johns Hopkins Hospital in the 1950s to stark white laboratories with freezers full of HeLa cells; from Henriettas small, dying hometown of Clover, Virginia — a land of wooden slave quarters, faith healings, and voodoo — to East Baltimore today, where her children and grandchildren live, and struggle with the legacy of her cells. Henriettas family did not learn of her immortality until more than twenty years after her death, when scientists began using her husband and children in research without informed consent. The story of the Lacks family — past and present — is inextricably connected to the dark history of experimentation on African Americans, the birth of bioethics, and the legal battles over whether we control the stuff we are made of. More information at rebeccaskloot.com.

“Skloot’s book is wonderful, deeply felt, gracefully written, sharply reported.” — Susan Orlean, author of The Orchid Thief

“This is an extraordinary book, haunting and beautifully told.” — ERIC SCHLOSSER, author of Fast Food Nation

When: Monday, February 1 2010, 07:00 PM
Where: McNally Jackson Books, 52 Prince Street, New York, NY, 1001
Full nationwide tour schedule and details