Solid stress facilitates spheroid formation: potential involvement of hyaluronan.

When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.

Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors.

The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor-host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.

A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins.

A general method for mapping tertiary interactions in membrane proteins using the visual pigment rhodopsin as a model is presented. In this approach, the protein is first assembled from two separately expressed gene fragments encoding nonoverlapping segments of the full-length polypeptide. Cys residues are then introduced into each of the two fragments such that juxtaposed residues are able to form disulfide cross-links in the protein either spontaneously or with the assistance of a Cu(2+)-(phenanthroline)3 oxidant. The cross-linked polypeptides are identified from a characteristic mobility shift on sodium dodecyl sulfate (SDS) gels as detected by Western blot analysis where the covalently bound heterodimer migrates with a mobility essentially identical to that of the native, full-length protein. Three different split rhodopsin mutants were prepared: one with a split in the loop connecting helices 3 and 4 (the 3/4 loop), one with a split in the 4/5 loop, and one with a split in the 5/6 loop. Each of these proteins when purified from transfected COS cells bound 11-cis-retinal, had a native absorption maximum at 500 nm, and activated transducin in a light-dependent manner. The cross-linking assay was tested with the rhodopsin mutant split in the 5/6 loop using the rho-1D4 antibody (which recognizes the carboxy terminal eight amino acids of rhodopsin) to detect the proteins on Western blots of SDS gels. Cys residues were substituted for Val-204 in the amino terminal fragment and Phe-276 in the carboxy terminal fragment of the rhodopsin mutant because Schwartz and co-workers [Elling et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeted delivery of DNA using YEE(GalNAcAH)3, a synthetic glycopeptide ligand for the asialoglycoprotein receptor.

In vivo gene therapy shows promise as a treatment for both genetic and acquired disorders. The hepatic asialoglycoprotein receptor (ASGPr) binds asialoorosomucoid-polylysine-DNA (ASOR-PL-DNA) complexes and allows targeted delivery to hepatocytes. The tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl) glutamate [YEE(GalNAcAH)3] has been previously reported to have subnanomolar affinity for the ASGPr. We have used an iodinated derivative of YEE(GalNAcAH)3 linked to polylysine and complexed to the luciferase gene (pCMV-Luc) in receptor-binding experiments to establish the feasibility of substituting ASOR with the synthetic glycopeptide for gene therapy. Scatchard analyses revealed similar Kd values for both ASOR and the glycopeptide. Binding and internalization of 125I-Suc-YEE(GalNAcAH)3 were competitively inhibited with either unlabeled ASOR or glycopeptide. The reverse was also true; 125I-ASOR binding was competed with unlabeled YEE(GalNAcAH)3 suggesting specific binding to the ASGPr by both compounds. Examination of in vivo delivery revealed that the 125I-labeled glycopeptide complex mimicked previous results observed with 125I-ASOR-PL-DNA. CPM in the liver accounted for 96% of the radioactivity recovered from the five major organs (liver, spleen, kidney, heart, and lungs). Cryoautoradiography displayed iodinated glycopeptide complex bound preferentially to hepatocytes rather than nonparenchymal cells. In vitro, as well as in vivo, transfections using the glycopeptide-polylysine-pCMV-luciferase gene complex (YG3-PL-Luc) resulted in expression of the gene product. These data demonstrate that the YEE(GalNAcAH)3 synthetic glycopeptide can be used as a ligand in targeted delivery of DNA to the liver-specific ASGPr.

Preparation of asialoorosomucoid-polylysine conjugates.

Asialoorosomucoid-polylysine (ASOR-PL) conjugates have been recently developed as carriers of electrostatically bound DNA for targeted delivery to the hepatic asialoglycoprotein receptor (ASGPr) for gene therapy. Using acid-urea gel electrophoresis we have found that previously reported procedures for the fractionation of ASOR-PL conjugates do not efficiently remove noncovalently bound polylysine (PL) from ASOR-PL. DNA complexes prepared with these conjugates have low solubilities, which limits their usefulness for subsequent experimentation, particularly in vivo. For ASOR-PL made by carbodiimide-mediated crosslinking with 5-kDa PL, dialysis against 1 M guanidine hydrochloride is effective to remove the low molecular weight unbound PL. Dialysis is not feasible when using higher molecular weight PLs, but preparative elution acid-urea gel electrophoresis was used to isolate crude ASOR-PL fractions free of unbound PL. ASOR-PL freed of PL by dialysis or electrophoresis was further fractionated by cation-exchange HPLC on carboxymethyl-functionalized columns eluted with a mixed pH-salt gradient. Early-eluting ASOR-PL fractions isolated by a combination of preparative elution acid-urea gel electrophoresis and cation-exchange HPLC were found to be preferred for the formation of soluble DNA complexes.