John M. Chirgwin

Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.

Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-beta (TGF-beta), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-beta inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-beta responses, any role of TGF-beta in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-beta promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant-negative mutant (TbetaRIIDeltacyt) of the TGF-beta type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-beta. In a murine model of bone metastases, expression of TbetaRIIDeltacyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant-negative signaling blockade by expression of a constitutively active TGF-beta type I receptor in the breast cancer cells increased tumor production of parathyroid hormone-related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant-negative TbetaRIIDeltacyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-beta in the development of breast cancer metastasis to bone, via the TGF-beta receptor-mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

Synthetic oligonucleotides were used to screen a rat striatal cDNA library for sequences corresponding to the tachykinin peptides substance P and neurokinin A. The cDNA library was constructed from RNA isolated from the rostral portion of the rat corpus striatum, the site of striatonigral cell bodies. Two types of cDNAs were isolated and defined by restriction enzyme analysis and DNA sequencing to encode both substance P and neurokinin A. The two predicted preprotachykinin protein precursors (130 and 115 amino acids in length) differ from each other by a pentadecapeptide sequence between the two tachykinin sequences, and both precursors possess appropriate processing signals for substance P and neurokinin A production. The presence of a third preprotachykinin mRNA of minor abundance in rat striatum was established by S1 nuclease protection experiments. This mRNA encodes a preprotachykinin of 112 amino acids containing substance P but not neurokinin A. These three mRNAs are derived from one rat gene as a result of differential RNA processing; thus, this RNA processing pattern further increases the diversity of products that can be generated from the preprotachykinin gene.