Probing Transcriptional Action in Human Tumor Cell Lines

At different ages and stages, our bodies use a complex array of signaling factors to inhibit or excite the growth of various cells. If these factors malfunction, however, the resulting acceleration of cell growth can lead to many forms of cancer. Of course, this also suggests an opportunity for researchers to develop treatments that would activate certain signaling factors and perhaps inhibit tumor growth.

One signaling factor of particular interest is transforming growth factor-beta (TGF-ß). This multifunctional polypeptide regulates a variety of cellular activities including proliferation, differentiation, apoptosis and expression of extracellular matrix proteins. TGF-ß is widely studied as the prototype for a large superfamily of growth and differentiation factors, and is perhaps best known for its potent ability to arrest growth in many cell types, thereby acting as a tumor suppressor.

Loss of TGF-ß-mediated growth inhibition is frequently correlated with tumor progression in various human cancers. To better understand this mechanism, scientists are trying to elucidate why tumors cells selectively alter this signal transduction pathway. An important first step towards this goal is to understand, at the transcriptional level, how TGF-ß controls gene expression.

Relating TGF-ß, proteins and cancer

Members of the TGF-ß superfamily signal through a unique set of serine-threonine kinase receptors. After TGF-ß (or a member of its superfamily) binds with a functional receptor complex, it triggers a cascade of intracellular signaling that can ultimately alter gene expression.

TGF-ß signaling is carried out, in part, by a group of intracellular proteins known as the Smads. Disruption of the Smad signaling complex often leads to tumor formation. For example, one of the Smad proteins, Smad4, is inactivated in a variety of cancers. Smad4 is often called a "common-partner Smad" or "co-Smad" because it is found in different Smad signaling complexes. This common partner is inactivated in more than 50% of pancreatic tumor cells and in 15% of colorectal cancers. It has also been correlated with malignant progression in colon, breast and other tissues.

Recently, Dr. Xuedong Liu and his research group at the University of Colorado in Boulder set out to investigate the consequences of Smad4 inactivation on the TGF-ß-controlled transcription program in tumor cells. Working in collaboration with scientists from Agilent, the Colorado team wanted to find out whether Smad4 is absolutely required for transmitting TGF-ß signals and whether loss of Smad4 causes similar alterations in gene expression in different types of tumor cells.

Analyzing Smad4-independent transcription

Agilent DNA microarrays

The research team used DNA microarrays to profile TFG-ß-regulated gene expression in four different cell lines that do not express Smad4. Two of these Smad4-null cell lines originated from pancreatic cancer cells and one cell line each originated from colon and breast cancer cells. Agilent's Human 1 cDNA microarrays and Human 1A Oligo microarrays were used to compare transcription profiles of these Smad4-null tumor cell lines before and after treatment with TGF-ß.

In these studies, TGF-ß treatment resulted in the up- and down-regulation of gene sets in all four Smad4-null cell lines, indicating that TGF-ß can alter gene expression via Smad4-independent mechanisms. Although Smad4 is a common partner in different Smad signaling complexes—and is indisputably involved in TGF-ß signaling under normal circumstances—it is not absolutely required for relaying TGF-ß signals from the cytoplasm to the nucleus.

The studies also revealed that the four different cell lines share a common set of eight Smad4-independent, TGF-ß-inducible genes. This suggests that inactivation of Smad4 may alter transcriptional programs of the TGF-ß signal, but that Smad4 itself is not absolutely required for TGF-ß signaling from cell surface to nucleus.

Looking to the future

Although the data obtained using Agilent's microarrays have yielded numerous insights, this is just the beginning of new, comprehensive studies. Future plans include the engineering of Smad4-deficient cell lines to re-express the normal Smad4 gene, as well as additional microarray analyses to pinpoint the Smad4-dependent and Smad4-independent genes. Ultimately, the Colorado team hopes to unravel the molecular details of TGF-ß signaling networks and to characterize the aberrant gene expression profiles occurring in tumor cells that harbor defective TGF-ß transcriptional programs.

For more information

Detailed discussions of this research are available as a poster brief and on the Customer-focused Articles page in the DNA Microarrays & Scanner section of our Web site. For additional information about Agilent's full range of products and resources, please go to the Life Sciences/Chemical Analysis main page.