Scientists at the Johns Hopkins Kimmel Cancer Center said they have preliminary evidence in laboratory-grown, human airway cells that a condensed form of cigarette smoke triggers epigenetic changes in the cells consistent with the earliest steps toward lung cancer development.

“Our study suggests that epigenetic changes to cells treated with cigarette smoke sensitize airway cells to genetic mutations known to cause lung cancers,” said Stephen Baylin, MD, the Virginia and DK Ludwig Professor for Cancer Research and professor of oncology at the Johns Hopkins Kimmel Cancer Center.

Details of the scientists’ experiments were described in the Sept. 11 issue of the journal Cancer Cell.

Baylin, co-corresponding author and Johns Hopkins scientist Hari Easwaran, PhD, and Michelle Vaz, PhD, Johns Hopkins postdoctoral researcher and first author on the study, suspected that the interplay of epigenetic and genetic changes may occur when normal lung cells develop into cancer, but, Baylin said, the timing of such changes was unknown.

To create the effect of tobacco smoke on cells, Vaz, Easwaran, Baylin and their colleagues began their studies with human bronchial cells, which line the airways of the lungs, and grew them in a laboratory. Every day for 15 months, the scientists bathed the cells with a liquid form of cigarette smoke, which they said is comparable to smoking one to two packs of cigarettes daily.

The scientists recorded the molecular and genetic changes in the smoke-exposed cells over 10 to 15 months, which the scientists said may be similar to 20 to 30 years of smoking, and compared the changes to bronchial cells that had not been exposed to the liquid smoke.

After 10 days of smoke exposure, the scientists found an overall increase in DNA damage responses to reactive oxygen species within the cells.

Between 10 days and three months, the cells exposed to smoke had a two- to four-fold increase in the amount of an enzyme called EZH2, which works to dampen the expression of genes. Baylin and other scientists have shown that EZH2 and its effects can precede abnormal DNA methylation in gene start sites.

After EZH2 enzymes rise, their levels taper off, and then, the scientists found two to three-fold increases in a protein called DNMT1, which maintains DNA methylation in the “start” location of a variety of tumor suppressor genes that normally suppress cell growth. When these genes are silenced a barrier is removed that might otherwise stop the cells from growing uncontrollably.

A host of other genes, which control many other cellular processes do not show such abnormal DNA methylation after smoke exposure.

Baylin said certain genes that control cell growth get turned down periodically during certain stages of life, including embryogenesis, when organisms are growing and developing rapidly. These genes can normally be turned on when cells need to stop growth and allow cells to mature. Chronic cigarette smoke exposure tends to block these cell maturation genes from properly turning on, said Baylin.

At the end of six months, the amount of EZH2 and DNMT1 enzymes had tapered off in the cells exposed to the smoke. However, the impact of the two methylation-regulating enzymes was still seen at 10 to 15 months, when scientists found decreased expression of hundreds of genes — many of which are key tumor suppressor genes such as BMP3, SFRP2 and GATA4 — in the smoke-exposed cells and a five- or-more-fold increase in the signaling of the KRAS oncogene that is known to be mutated in smoking-related lung cancers.

However, no mutations were found in the KRAS gene itself or the tumor suppressor genes during the 15-month period of cigarette smoke exposure. These abnormally methylated and silenced genes, said Baylin, would have blocked the increase in KRAS signaling if the genes had been properly activated under smoke-free circumstances.