2163T_web_25-1 4/20/07 11:49 AM Page 1 TEAM-B 101:WHQY013:whLod6:Web Exp: C L A S S I C E X P E R I M E N T 2 5 . 1
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2163T_web_25-1 4/20/07 11:49 AM Page 1 TEAM-B 101:WHQY013:whLod6:Web Exp:
C L A S S I C
E X P E R I M E N T
2 5 . 1
STUDYING THE TRANSFORMATION OF CELLS BY DNA TUMOR VIRUSES Sambrook et al., Proc. Nat’l. Acad. Sci. USA, 1968, 59:1290 and 1294
Not many diseases have spurred scientists to research and understand their causes more than have human cancers. Scientists recognized long ago that some viruses could induce tumors in animals, and in the 1960s and 1970s, research in this field surged. One of the pioneers in the field was Renalto Dulbecco, who, in 1968, reported that a tumor-causing virus could insert its DNA into the genome of the cell it transformed.
Background Understanding the molecular and cellular events that occur in cancer has long been a goal of biological research. Scientists have sought to understand how a normal cell becomes transformed into a tumor cell. Although they recognized early in the last century that some viruses, such as Rous sarcoma virus (RSV), could induce tumors in animals, it wasn’t until many years later that they investigated the viral causes of cancer in depth. Such investigation was aided by the development of techniques to study viruses in cultured cells rather than in living organisms. In the late 1950s, Dulbecco adapted a number of techniques used to manipulate bacteriophages—viruses that infect prokaryotic cells—for use in studying animal viruses in cultured eukaryotic cells. He then turned his attention to studying cell transformation by viruses in cultured cells, using the DNA tumor viruses simian virus 40 (SV40) and polyoma virus. Both SV40 and polyoma viruses could infect a number of different types of cells in culture. Cells that are susceptible to infection by these viruses fall into two classes: permissive cells, which produce more virus after infection, and nonpermissive cells, which
do not. Whereas nonpermissive cell lines did not produce new virus after infection, these were the only cells that the tumor viruses could transform. Something was happening in the nonpermissive cell lines that caused them to be transformed, rather than produce new virus. Dulbecco noted that a similar phenomenon had been observed in bacteriophage infections. Some phages cause the rapid production of new phages in a lytic infection (see Figure 4-46 in the text). Other phages— through the process of lysogeny—lay dormant in the infected cell, while its DNA becomes integrated into the bacterial genome (Chapter 4). Dulbecco wondered if viral infection in nonpermissive cells might be similar to lysogenous phage infection in bacteria. He and others demonstrated that the viral DNA could be detected in cells transformed by polyoma or SV40 viruses. Could this viral DNA be integrated into the cellular DNA? In the late 1960s, Dulbecco set out to find the answer.
The Experiment To determine the state of SV40 DNA in transformed cells, Dulbecco used nucleic acid hybridization, which is a powerful technique that can detect a relatively small DNA sequence within a larger sample of DNA. In his experiment, Dulbecco isolated viral DNA from purified SV40 particles. He then used purified RNA polymerase to transcribe the viral DNA into RNA in vitro. He labeled the RNA radioactively by including [3H]cytosine triphosphate (CTP) in the in vitro transcription reaction, creating a radioactively labeled probe. Next, he prepared genomic DNA from SV40-transformed cells, heated it to separate the double-stranded
DNA into two single-stranded molecules, and then bound single-stranded DNA to nitrocellulose filters. He then treated the single-stranded DNA bound to the filters with the radioactively labeled RNA probe. Through basepairing interactions, the radioactively labeled RNA specifically hybridized to the SV40 DNA. After washing the filters to remove unhybridized RNA, he analyzed the filters by scintillation counting. Under optimal conditions, radioactivity on the membranes correlates with the presence of SV40 DNA in the sample. Indeed, Dulbecco successfully employed this technique to detect SV40 DNA in the transformed cells. Once he determined that the viral DNA was present in the transformed cells, Dulbecco proceeded to determine the form and the location of SV40 DNA in a transformed cell. To do this, he took advantage of physical differences between the SV40 viral DNA and the genomic DNA isolated from the infected 3T3 cells (SV3T3). SV40 DNA isolated from viruses is in a circular supercoiled form (see Figure 4-8 in the text), which can be separated from SV3T3 genomic DNA by equilibrium density centrifugation through a gradient of cesium chloride (CsCl) in the presence of ethidium bromide. Ethidium bromide intercalates into linear DNA more readily than does circular DNA, changing its density and thus allowing the separation of supercoiled viral DNA from linear genomic DNA. As a control, Dulbecco performed the same analysis on polyoma virus–transformed 3T3 cells (Py3T3) (like SV40, polyoma DNA isolated from viruses is circular and supercoiled). He immobilized DNA isolated by these procedures onto nitrocellulose filters and hybridized the radioactively labeled
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Demonstration that SV40 DNA Is Integrated in Transformed Cells
A. C S C L CENTRIFUGATION Cells
Circular DNA (CPM)
Linear DNA (CPM)
SV3T3
69 5
358 2
Py3T3
64 7
129 12
Py3T3 SV40 viral DNA
385 11
198 5
B. ALKALINE SUCROSE GRADIENTS Cells
CPM hybridized
SV3T3
620 12
Py3T3
248 2
CPM Counts per minute. [Data adapted from Sambrook et al., Proc. Natl. Acad. Sci USA, 1968, 59:1290 and 1294.]
SV40 RNA. He found that the SV40 RNA hybridized only to the linear fraction of DNA from the SV3T3 cells (see Table, A). The level of hybridization to the supercoiled DNA was no greater in the SV3T3 cells than it was in the Py3T3 cells, indicating that any radioactivity detected in this fraction was at background levels. As an additional control, Dulbecco added supercoiled DNA isolated from SV40 to the Py3T3 cell extract. In these cells, he could detect the DNA specifically in the supercoiled DNA fraction of the DNA isolated from these Py3T3 cells, and not in the linear DNA fraction (see Table, A). From these experiments, he concluded that the SV40 DNA was not in its supercoiled, viral form in transformed cells. To assure that the SV40 DNA in the linear fraction was part of the SV3T3 genomic DNA, and not linearized SV40 viral DNA, Dulbecco performed alkaline sucrose gradients. He layered the cells onto the alkaline gradient, then incubated them to allow the cells to break open. This
procedure minimized inadvertent mechanical shearing of cellular DNA. Once lysis was complete, the cellular DNA was sedimented in the sucrose gradient. Dulbecco then compared the hybridization of SV40 RNA to DNA isolated from SV3T3 and Py3T3 and found that it specifically hybridized to the SV3T3 cells (see Table, B). Because the SV40 DNA always hybridized to the high-molecularweight genomic DNA, he concluded that it was covalently attached to the cellular DNA.
Discussion Dulbecco’s demonstration that the SV40 DNA was integrated into the genome of transformed cells began a new wave of thinking about cellular transformation. With the advent of restriction endonucleases, as well as other molecular-biology techniques, scientists demonstrated that SV40 integrates into random sites in the host cell genome. Researchers have found that a number of other tumor-inducing DNA
viruses, as well as RNA-based retroviruses, integrate into the host cell genome. They later showed that viral integration can contribute to tumor formation by disregulating the expression of key cellular genes involved in cell growth, or by allowing expression of viral proteins that derail normal cellular mechanisms to inappropriately stimulate cell growth and proliferation. Although tumor viruses are more prevalent in mice and other animals, several human tumor viruses, such as human T-cell leukemia/lymphoma virus (HTLV) and human papilloma virus (HPV), are known to integrate into the host cell genome and can lead to cancer. By inspecting the sites of viral insertion, scientists have discovered a variety of oncogenes in work predicated on Dulbecco’s initial studies on DNA tumor viruses and their ability to integrate into the host genome. In 1975, the Nobel Prize in Physiology or Medicine was awarded to Dulbecco for his vast contributions to this field.