Bloom syndrome (BS) is an autosomal recessive disorder characterized by small stature, sun-sensitivity, immunodeficiency, male infertility, increased frequency of diabetes, and a predisposition to a variety of cancers at an early onset. Cells from affected individuals exhibit genomic instability including chromosome breakage, an excess of somatic mutations and elevated levels of sister chromatid exchanges (SCE’s). Bloom syndrome occurs most frequently in the Ashkenazi Jewish population with patients almost exclusively homozygous for a frameshift mutation resulting from a 6 bp deletion/7 bp insertion at nucleotide 2,281 (BLMAsh). This mutation causes premature termination of the encoded gene product producing a truncated protein of 739 amino acids while the full length protein contains 1417 amino acids.
Figure 1-Figure 1. RT-PCR analysis of BLM mRNA in WI-38, HeLa and LAI-5S cell lines. A.) RT-PCR products generated with primers located in exons 19 (nucleotides 3804-3823 of Accession number NM_000057) and 22 (nucleotides 4221-4202 of Accession number NM_000057) was performed using 60ng of WI-38, HeLa and LAI-5S total RNA. The first three lanes on the left show RT-PCR performed with an amplification step at 28 cycles and the last three lanes at 31 cycles. Lane (-) is a non-template control. The position and size of BLM is indicated by an arrow. A 100bp ladder was run on the gel for size comparison. B.) RT-PCR products generated with primers specific for β-actin (F: nucleotides 620-640 of Accession number BC001301; R: nucleotides 1064-1044 of Accession number BC001301) using 19 cycles of amplification. 60ng of RNA isolated from each of the cell lines was used. Lane (-) is a non-template control.
Figure 2-Figure 2. Sequence alignment of BLM. The sequenced BLM PCR product (BLM pr. BLM-R) was aligned with the BLM mRNA sequence from GenBank (nucleotides 3981- 4141 of NM_000057).
Figure 3-Figure 3. Western Blot analysis of the BLM protein. (A) Equivalent amounts of total cell protein (45µg) was loaded onto an 8% SDS polyacrylamide gel and transferred to nitrocellulose. A polyclonal anti-BLM antibody was used to probe the nitrocellulose. The position and size of the proteins recognized by the antibody is indicated with arrows. A prestained molecular weight marker (Bio Rad) was used to determine the sizes of the products. (B) The blot was also probed with an anti-GAPDH antibody to confirm equal protein loading. The position and size of GAPDH is indicated.
These studies revealed that the WI-38, HeLa and LAI-5S cell lines do not exhibit differential expression of the BLM mRNA; however, there appears to be variation in the expression of the protein. RT-PCR analysis showed equivalent amounts of mRNA expression in each of the three lines. DNA sequencing analysis confirmed that the RT-PCR product amplified was, in fact, a portion of the BLM mRNA sequence. Finally, Western blot analysis revealed the presence of the full-length BLM protein in the WI-38 cells but not in the HeLa or LAI-5S cells. In addition, a smaller, immunologically-related protein of ~ 60kD was observed in all three cell lines but appears to be expressed to a greater extent in the two cancer cell lines. It is possible that the smaller molecular weight protein represents an alternatively spliced product of the BLM gene. The expression pattern of both the BLM and the smaller molecular weight protein lead us to speculate that the cancer cell lines may exhibit a higher degree of alternative splicing of the BLM gene product.
Additional studies should focus on characterizing the smaller molecular weight protein in order to determine if alternative splicing of the BLM gene product occurs. It is possible that this protein is unrelated to the BLM protein but it awaits further analysis using a monoclonal antibody in order to determine if it is specifically associated with BLM. Furthermore, more detailed experiments examining other cancer-derived cell lines would help ascertain if a relationship exists between the expression of BLM and tumorigenesis. Because alternative splicing has a strong role in the development of cancer, splicing events can be seen as a potential target in the search for a possible therapy for cancer patients.
I would like to thank Dr. Rubin for his guidance and the opportunity to pursue this project along with Brian Fox and Jinsong Qiu for their extreme patience and time. I would also like to acknowledge Dr. Robert Ross for generously providing LAI-5S cells.
|This document was last modified 01/31/2006.|
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