David Waltregny

Gene transcript quantitation by real-time RT-PCR in cells selected by immunohistochemistry-laser capture microdissection.

Studying tissue-based gene expression in different cell populations often requires immunohistochemistry-guided microdissection. However, mRNA degradation occurs during long staining procedures. We combined a novel rapid immunoperoxidase technique with laser capture microdissection (LCM) and real-time quantitative RT-PCR to compare p27 mRNA expression in prostatic basal/secretory cells. Eight frozen prostate sections were immunostained with antibody 34&bgr;E12 (high–molecular-weight keratin). Secretory and basal cells were separately collected by LCM. p27 transcripts from each cell group were quantitated by real-time RT-PCR, with GAPDH as standard. Immunostaining took 22 minutes, with RNA extraction from ∼40 dissected cells from each compartment initiated within 40 minutes. Qualitative RT-PCR gave a product of the expected size from each sample. Quantitative RT-PCR gave basal/secretory p27/GAPDH ratios of 0.99–16.24 (mean 5.53 ± 0.643). Immunostaining for keratin 34&bgr;E12 can be done on frozen sections in ∼20 minutes, and mRNA from pure cell populations can be quantitated by RT-PCR. We used this technique to show that p27 transcript levels are greater in basal than in secretory prostate cells, suggesting, when combined with prior studies, that regulation of p27 occurs at the protein level in normal cells. This technique may have wide applicability to studies of gene expression in distinct cell populations in heterogeneous tissues.

Detection of Bone Sialoprotein in Human (Pre)neoplastic Lesions of the Uterine Cervix

Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in the mineral compartment of developing bones. BSP is detected in a variety of human cancers, particularly those that metastasize to the skeleton. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. Since squamous cell carcinoma (SCCs) of the uterine cervix also frequently metastasizes to bone, we investigated whether BSP is expressed in human cervical cancer. We examined BSP expression in cervical tissue samples from 47 patients, including 19 normal tissues, 20 squamous intraepithelial lesions (SILs) (9 low and 11 high grade) and 8 invasive SCCs. BSP protein expression was evaluated by the immunophosphatase technique using a BSP polyclonal antibody in paraffin-embedded cervical biopsies. The abundance of BSP protein was significantly higher in invasive SCCs and high grade SILs than in normal cervix tissue samples and low grade SILs, which showed no or a low level of anti-BSP immunoreactivity. In situ hybridization experiments performed on representative cervix invasive SCCs frozen sections revealed that BSP transcripts were detectable in these lesions. Our study demonstrates that BSP expression is a common feature in high grade SILs and invasive SCCs of the uterine cervix. The prognostic value of BSP detection in these lesions and the potential role of BSP as an angiogenic factor in this type of cancer are currently under investigation.