Overview of expression of matrix metalloproteinases (MMP-17, MMP-18, and MMP-20) in cultured human cells.

We recently described the cell type distribution of several matrix metalloproteinases (MMP-1 through MMP-16). In this report we extend this study by analysis of three recently described MMPs. PCR primers for MMP-17, MMP-18, and MMP-20 were optimized for use in RT-PCR. The results demonstrate one or more cell lines or tissue that express mRNA for each of these newly described MMPs.

Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells.

Matrix metalloproteinase-8 (MMP-8) is a neutral metalloproteinase of the fibrillar collagenase family that also includes MMP-1 and MMP-13. In contrast to the other collagenases, MMP-8 has a very limited tissue distribution, thought to be restricted to neutrophils and chondrocytes. In a previous study, we observed MMP-8 expression in human melanoma cells. This observation led us to assess in more detail the expression of MMP-8 in normal and malignant melanocytic cells. We found that MMP-8 was expressed by 11 out of 12 human melanoma cell lines tested and all 10 primary melanomas we examined, but was not expressed by four primary neonatal melanocyte strains. Since melanocytes arise from highly motile neural crest cells, we examined the hypothesis that MMP-8 might be expressed by neural crest cells. RT-PCR analysis of post-implantation mouse embryos indicated the presence of MMP-8 transcripts at E9.5. In situ hybridization and immunohistochemistry of mouse embryos between E9.5-E14.5 demonstrated MMP-8 expression in the surface ectoderm, neural crest cells and chondrocytes. MMP-8 was also detected in neural crest cell migration located in the circumference of the neural tube, branchial arches and the notochord. In addition, MMP-8 expression was observed between the somites, in circumscriptive areas of the developing brain, heart, and eye, and in the interdigital zones of the limbs. In summary, we found MMP-8 to be the first fibrillar collagenase expressed during development. In contrast to its restricted tissue expression post-partum, MMP-8 was present in multiple embryonic tissues, including neural crest cells. The production of MMP-8 by migrating neural crest cells may contribute to their ability to degrade fibrillar collagen matrices while in transit.

Overview of matrix metalloproteinase expression in cultured human cells.

The matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis both by immunohistochemical studies and from the observation that specific metalloproteinase inhibitors block tumor invasion and metastasis. Oligonucleotide primers for thirteen MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16) were optimized for use in RT-PCR. A semi-quantitative RT-PCR assay was used to determine the pattern of MMP mRNA expression in 84 normal and transformed or carcinogen transformed human cell lines and strains derived from different tissues. The results demonstrate one or more cell lines which express thirteen members of the MMP family. In addition, various oncogene transfected human fibroblast cell strains were analyzed for MMP expression. We confirm that over-expression of the H-ras oncoprotein correlates with up-regulation of MMP-9 and demonstrate that over-expression of v-sis also up-regulates MMP-9. A cell line immortalized following myc expression was found to up-regulate MMP-7, MMP-11 and MMP-13. Inappropriate expression of several MMP mRNAs was detected in breast, prostate, bone, colon and oral tumor derived cell lines. Identification of at least one cell line expressing each of thirteen MMPs and the observation of oncogene induced expression of several MMPs should facilitate analysis of the transcriptional mechanisms controlling each MMP.

RT-PCR without RNA isolation.

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.

The efficacy of cryopreserved hamster ova in the sperm penetration assay.

To solve the logistical problems of the sperm penetration assay (SPA) to provide just a sufficient number of hamster ova exactly when they are needed, a new method to cryopreserve the ova has been devised. The ova, suspended in a 1.5 M solution of propylene glycol as a cryoprotectant in an isotonic salt solution, were frozen in 1/4 mL plastic straws. Included in each straw was a sucrose solution, isosmotic to the propylene glycol solution, to serve as an osmotic buffer during dilution of the cryoprotectant out of the ova. This one-step method of dilution permitted the ova to be recovered and diluted out of the cryoprotectant within the straw in which they had been originally frozen. A total of 547 cryopreserved ova were thawed, 504 (92.1%) of which were morphologically normal after they had been incubated at 37 degrees C for 3 hours. After removal of the zonae, the frozen-thawed ova were compared with fresh, control ova in SPAs of donor and patient semen that had been capacitated in TEST-yolk buffer. The percent penetration and penetration index of fresh versus cryopreserved ova did not differ significantly for either donor or patient semen.

Use of oxygen-permeable silicone rubber pouches for growing mass cultures of bacteria.

Growth of most bacteria often involves the use of expensive incubated shaker systems. In this report, oxygen-permeable silicone rubber pouches, with oxygen permeability over 100 times higher than other polymers, were employed for growing bacterial cultures. With little, if any, agitation oxygen-permeable silicone rubber pouches produced bacterial growth rates equivalent to growth rates obtained in shaker flasks. The silicone rubber pouch described has a glass cuvette integrated into its design that permits readings of bacterial density without opening the pouch. One can sterilize and store powdered bacterial culture medium in silicone rubber pouches; therefore, bacterial cultures can be initiated by simply adding water and bacteria.

Definition of a tumor suppressor locus within human chromosome 3p21-p22.

Cytogenetic abnormalities and high-frequency allele losses involving the short arm of human chromosome 3 have been identified in a variety of histologically different neoplasms. These findings suggest that a tumor-suppressor gene or genes may be located in the region of 3p14-p25, although there has been no definitive functional proof for the involvement of a particular region of 3p. We report a rapid genetic assay system that has allowed functional analysis of defined regions of 3p in the suppression of tumorigenicity in vivo. Interspecific microcell hybrids containing fragments of chromosome 3p were constructed and screened for tumorigenicity in athymic nude mice. Hybrid clones were obtained that showed a dramatic tumor suppression and contained a 2-megabase fragment of human chromosomal material encompassing the region 3p21 near the interface with 3p22. With these hybrid clones, we have defined a genetic locus at 3p21-p22 intimately involved in tumor suppression.

Extraction of RNA from single frozen sections.

The sensitivity of the reverse transcriptase-polymerase chain reaction (RT-PCR) makes it ideally suited for the detection of changes in gene expression. Unfortunately, traditional methods for RNA isolation require time-consuming procedures that are not appropriate for small samples, such as individual frozen sections. This report describes a new technique that permits the rapid extraction of RNA from individual frozen histological sections. RNA is extracted by incubating a frozen section in an RT-PCR compatible buffer solution containing RNase inhibitor and dithiothreitol. RNA isolated from frozen sections is stable at room temperature for up to 3 h under the conditions described. Alternatively, extracts can be frozen for later use. When maintained in a dry state at room temperature, RNA in sections remained stable for 2 weeks. Histological, immunohistochemical, or in situ analyses can be carried out with sections that are immediately adjacent to those used for extracting RNA. The simplicity and economy of this procedure may foster the development of prognostic screens that can be performed in parallel with traditional histopathological analysis.

Organization and expression of human telomere repeat binding factor genes.

The ends of mammalian chromosomes terminate in structures called telomeres. Recently a human telomere repeat binding factor (TRF1) that binds the vertebrate TTAGGG telomeric repeat in situ was isolated by Chong et al. (1). TRF1 regulates telomere length (2), which is often altered in cancer cells. To understand their genetic organization, TRF1 genes were localized to human chromosomes 13cen, 21cen, and Xq13 by analysis of human monochromosomal hybrids, and by fluorescent in situ hybridization. We also confirmed the recent localization of a human TRF1 gene to chromosome 8, and provide evidence that this locus is alternatively spliced. In contrast to the TRF1 genes on chromosomes 8 and X, the chromosomes 13 and 21 TRF1 genes contained a 60 bp deletion in the coding region. The results suggest that two distinct forms of TRF1 are expressed and that the TRF1 gene family includes at least three pseudogenes whose dispersal in the human genome may have occurred via cDNA intermediates.

Chromosomal sublocalization of the transcribed human telomere repeat binding factor 2 gene and comparative mapping in the mouse.

Telomere repeat binding factor 2 (TERF2) is one of two recently cloned mammalian telomere binding protein genes. TERF2 binds as a dimer with high affinity to the double-stranded TTAGGG telomeric repeat through an evolutionarily conserved myb-type DNA binding domain. TERF2 prevents telomere end-to-end fusion and may be important in maintaining genomic stability. We localized the transcribed TERF2 gene to human chromosome 16q22.1, tightly linked to the EST HUM000S343. The mouse Terf2 gene is situated by itself in a newly defined "bin" on chromosome 8 one crossover distal to Psm10 and Sntb2. Human TERF2 and mouse Terf2 are therefore part of a large evolutionarily conserved linkage group comprised of at least 25 known paralogous genes between human chromosome 16q and mouse chromosome 8.