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.

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 212: Apocalypse TWiV 122112 212

On episode #212 of the science show This Week in Virology, the TWiVerers answer listener email about genetically modified chickens, a hendra vaccine for horses, online education, curing color blindness, Roosevelt and polio, Th cells, and much more.

You can find TWiV #212 at www.microbe.tv/twiv.

TWiV 197: Cloning HeLa cells with Professor Philip I Marcus

On episode #197 of the science show This Week in Virology, Vincent travels to the University of Connecticut to meet up with Professor Philip I. Marcus to discuss his development of the single cell cloning technique in the early 1950s.

You can find TWiV #197 at www.microbe.tv/twiv.

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

Rhinovirus and zinc part 5: Magnesium is not the culprit

If you have been following the results of my experiments on inhibition of rhinovirus replication by ZnCl2, you know that I’ve been trying to determine why concentrations of the salt higher than 0.1 mM are toxic to HeLa cells. I have found that 0.1 mM ZnCl2 does inhibit rhinovirus plaque formation but not sufficiently to be able to select resistant mutants. In today’s set of experiments I asked whether the presence of MgCl2 in the agar overlay potentiates zinc toxicity.

We always include MgCl2 (40 mM) in the agar overlay when assaying rhinoviruses, because it significantly improves plaque size. The following monolayers of HeLa cells were inoculated with 200 plaque-forming units of rhinovirus type 1a, then incubated at 32°C for 5 days. The effect of MgCl2 is remarkable.

hrv_mg_effect

The use of MgCl2 to improve plaque formation of certain picornaviruses dates to the 1962 observation that the salt increases susceptibility of cells to poliovirus infection. It was later shown to enhance plaque formation of rhinoviruses.

Unfortunately, omission of MgCl2 from the agar overlay has no effect on zinc toxicity. As shown below, cell viability was still poor in the presence of 0.2 mM ZnCl2.

hrv_mg_zn

What’s next? One reader suggested that I try selecting HeLa cells in successively higher concentrations of ZnCl2 to obtain cells resistant to the toxic effects of the salt. This approach is under way. I will also attempt to propagate rhinovirus in cells covered with liquid growth medium rather than under agar. If 0.1 mM ZnCl2 is included in the culture medium, a good fraction of the viruses produced might be resistant to the salt. The virus produced in these infected cells will be used to infect fresh cells, also in culture medium with ZnCl2. After 5-10 passages in this manner the majority of the viral population should be resistant to ZnCl2. This is a more time consuming approach that the plaque assay, but might yield zinc resistant rhinovirus mutants.

Wallis, C., & Melnick, J. (1962). Magnesium chloride enhancement of cell susceptibility to poliovirus. Virology, 16 (2), 122-132 DOI: 10.1016/0042-6822(62)90287-8

Fiala M, & Kenny GE (1966). Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium. Journal of bacteriology, 92 (6), 1710-5 PMID: 4289358

Rhinovirus and zinc part 4: cell toxicity

My experiments to understand how ZnCl2 inhibits rhinovirus replication have been thwarted by the finding that concentrations of the salt higher than 0.1 mM are toxic for cultured HeLa cells. The cells can tolerate 0.1 mM but not 0.2 mM ZnCl2. Last week I asked whether I could identify a concentration between 0.1 and 0.2 mM that does not harm the cells but inhibits viral plaque formation >99%. Here are the results.

hrv_zinc_4

Unfortunately even 0.125 mM ZnCl2 is toxic to the cells – which is surprising since the cells can tolerate 0.1 mM. The goal of these experiments is to identify Zn-resistant rhinovirus mutants, and this cannot be done with cell monolayers that are not healthy.

I have one more idea for how to get around the ZnCl2 toxicity. To improve the formation of rhinovirus plaques, 25 mM MgCl2 is added to the agar overlay. It is possible that this high amount of magnesium, together with 0.2 mM ZnCl2 or higher, is toxic to cells. Therefore I will determine whether Zn toxicity is reduced if MgCl2 is omitted from the agar overlay. The rhinovirus plaques will be smaller but that is a reasonable trade-off for healthier monolayers.

If omitting MgCl2 doesn’t work, then I will have to select Zn resistant rhinovirus mutants in cells propagated in liquid cell culture medium. I’ll explain that approach next week.

The amazing HeLa cells of Henrietta Lacks

spinner200One of the most widely used continuous cell lines for virology is the HeLa cell line, which was derived in 1951 from Henrietta Lacks. What is the origin of this amazing cell line?

In early 1951, Ms. Lacks, a 31-year old mother of five children, 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 provided to Dr. George Gey. He was head of tissue culture research at Hopkins who for years had been attempting to produce a line of immortal human cells.  When Ms. Lacks died in October 1951, Dr. Gey announced on national television that he had produced from Ms. Lacks’ tumor a line of cells that propagated as no other cells ever had before. He called them ‘HeLa’ cells in her honor, and showed a vial of the cells to the television audience.

HeLa cells have since been used in many laboratories all over the world to make countless  research discoveries. For example, shortly after Dr. Gey announced the HeLa cell line, it was used to propagate poliovirus, an event that played an important role in the development of poliovirus vaccines. But Ms. Lacks’ family never learned about the important cells that were derived from her until 24 years after her death. The history of this event, described in the Johns Hopkins Magazine, is a commentary on the lack of informed consent common in medical research at the time.

My laboratory uses HeLa cells for propagating and studying many different viruses. We have maintained the cells in suspension cultures (pictured) since 1983. Over the course of 26 years, 600,000,000 HeLa cells have been produced in my laboratory each week, for a total of 800 billion cells. That is a lot of cells, but it’s nothing compared with the total number of cells – approximately 100 trillion – that make up a human.