Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleoside methylphosphonate-neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the mouse.

Development of oligodeoxynucleotides (oligo-dNs) and their analogs as therapeutic agents is complicated by their low rate of transport across cellular membranes, which is required for interaction with the intracellular complementary nucleic acid sequences, and the lack of tissue-specific delivery. To overcome these obstacles, bioconjugates between cell surface receptor ligands and oligodeoxynucleoside methylphosphonates (oligo-MPs) have been constructed containing homogeneous, chemically defined covalent linkages. We have previously established that a model conjugate, [32P]-labeled [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7 (1), is delivered to Hep G2 cells in a ligand-specific manner, reaching a peak value of 26 pmol per 10(6) cells after 24 hours incubation at 37 degrees C (Hangeland et al., 1995). In this work, the in vivo behavior of this conjugate is explored. Administration of this conjugate to mice via tail vein injection demonstrates rapid uptake in liver to the extent of 69.9 +/- 9.9% of the injected dose after 15 minutes. Thereafter, the conjugate and its metabolites are rapidly cleared via the kidney and urine. Polyacrylamide gel electrophoresis analysis of extracts of Hep G2 cells and mouse liver reveal the conjugate 1 to be extensively metabolized. In contrast, the conjugate found in mouse urine is largely intact. These data show that this novel, biodegradable delivery vehicle represents a viable approach for the delivery of antisense oligo-MPs and other oligo-dN analogs to the liver for therapeutic and diagnostic applications.

Differential extracellular matrix gene expression by fibroblasts during their proliferative life span in vitro and at senescence.

Increasing evidence supports the idea that the finite proliferative life span of normal fibroblasts is a differentiation-like phenomenon. If this were correct, an ordered sequence of differential gene expression should be associated with the in vitro progression of cells from low passage to high passage (senescence). To define the pattern of expression of fibroblast differentiation-associated genes during this in vitro progression, we have determined the temporal pattern of expression of extracellular matrix (ECM) genes in Syrian hamster dermal fibroblasts as a function of passage level and percentage of proliferative life span in vitro. Steady-state mRNA levels were determined by Northern and dot blot analyses of total cellular RNA hybridized with cDNA probes specific for fibronectin, procollagen alpha 1III, and procollagen alpha 1I. Cells were analyzed at 24 hr postconfluence to minimize the presence of actively proliferating cells, and because maximal levels of fibronectin, alpha 1III, and alpha 1I mRNAs were observed 24 hr postconfluence. Unique, multiphasic patterns of expression of each of these ECM components were observed as the cells progressed from low passage to high passage. As the cells reached midhigh passage, fibronectin mRNA levels increased. This midpassage increase in fibronectin was followed by an increase in the level of alpha 1III mRNA as the cells reached the end of their in vitro proliferative life span, and then alpha 1I when the cells entered the postmitotic senescent phase, at which time the level of fibronectin mRNA also declined. A similar overlapping cascade pattern of up-regulation of these genes is seen during development and wound repair. This suggests that as cultured fibroblasts reach the end of their proliferative life span, they reinitiate a gene expression program used in tissue development and repair.

NMR studies of intracellular water at 300 MHz: T2-specific relaxation mechanisms in synchronized or EGF-stimulated cells.

Responses specific to the spin-spin relaxation time (T2) have been observed in two time-dependent studies of the intracellular water in normal and transformed Syrian hamster fetal fibroblasts. At 300-MHz (7.0 T), the spin-lattice relaxation time (T1) was insensitive to several aspects of cellular physiology that produced changes in the T2 and the apparent self-diffusion coefficient (Dapp) of intracellular water. In normal cells stimulated with epidermal growth factor (EGF), T1 was insensitive to time-dependent changes detected by T2 and Dapp. In synchronized tumor cells, T1 was insensitive to cell-cycle-dependent changes detected by T2. The strongly coupled behavior of T2 and Dapp that was observed as a function of time in EGF-stimulated cells indicates that the diffusion of intracellular water through inhomogeneous local magnetic field gradients produced effects observable in T2. Conformational changes in large intracellular macromolecular assemblies such as chromatin or the cytoskeleton may alter the magnitude and inhomogeneity of local field gradients, producing responses in T2 and Dapp only.

Age-related differences in promoter-induced extension of in vitro proliferative life span of Syrian hamster fibroblasts.

The proliferative capacity of Syrian hamster dermal fibroblasts has been previously shown to be inversely related to the age of the donor (Mech. Ageing and Dev., 34 (1986) 151). The present study demonstrates an inverse correlation between in vivo age and the in vitro morphological and proliferative response of Syrian hamster dermal fibroblasts to the tumor promoter phorbol-12,13-didecanoate. Treatment of fetal fibroblasts with promoter increased the proliferative life span of the cultures by approximately 2-fold, but did not increase the frequency of conversion to established cell lines. Neonatal and young adult fibroblasts exhibited intermediate responses to promoter treatment, showing 54.9% and 33.1% extension, respectively. In contrast, promoter treatment had no significant effect on aged adult fibroblasts. Maximal extension required continual treatment beginning in primary culture or at passage 1. Promoter-induced extension of proliferative life span appears to be mediated through the prolonged maintenance of small, highly proliferative cells that are present in primary cultures of these cells.

Studies of the T1 and T2 of intracellular water as a function of frequency in normal and transformed fetal cells.

Frequency-dependent values of the spin-lattice relaxation time (T1) and the spin-spin relaxation time (T2) have been obtained for intracellular water in normal and transformed Syrian hamster fetal fibroblasts. Values of T1 and T2 were obtained for normal and transformed cells at 24.3 (0.57 T), 100 (2.4 T), 300 (7.0 T), and 400 MHz (9.4 T). At each frequency, values of T1 were the same for both normal and transformed cells, whereas values of T2 were lower for one passage of transformed cells. As expected, T1 increased with frequency. However, T2 decreased with frequency for both normal and transformed cells. The frequency dependence of T2, was similar for all cells; thus, the ability of T2 to make a distinction between normal and transformed cells did not change with field.

Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts.

A longitudinal study was performed to confirm the inverse relationship between in vivo age and in vitro proliferative capacity previously observed in a cross-sectional study, and to investigate the relationship between the growth of dermal fibroblasts in vitro and a physiological function (i.e., wound repair) that is known to decline with age in vivo. Fibroblast cultures were generated from skin punch biopsies from 12 male hamsters beginning at 1 month of age and at 6-months interval thereafter until the natural death of the animal. All cultures from all individuals exhibited finite proliferative capacity, and an inverse relationship was observed between donor age and maximum in vitro proliferative capacity. In addition, a direct correlation between the in vitro proliferative capacity of the dermal fibroblasts in vitro and the repair efficiency of the biopsy site was observed. However, these changes in the in vitro proliferative capacity and in vivo wound repair efficiency were not progressive beyond 12-18 months of age and were not indicative at any age of an individual's ultimate lifespan. This study provides evidence that in vitro proliferative capacity of dermal fibroblasts and in vivo wound repair may be comparable phenomena that share a common mechanism. However, the nonprogressive nature and the lack of correlation between these phenomena and the individual's ultimate lifespan indicate that their use as biological markers of aging is limited to animals younger than the mean lifespan of the species.

Longevity and age-related pathology of LVG outbred golden Syrian hamsters (Mesocricetus auratus).

A colony of male Lakeview Golden (LVG) Syrian hamsters has been maintained for the last nine years as a source of various tissues for cellular aging studies. Observations on this colony also yielded data on survival time and physical and pathological manifestations of aging in this strain. Based on 150 spontaneous deaths, the median life span was found to be 19.5 months. The maximum life span was 36 months and the minimum 6 months. A cross-sectional pathological survey of sacrificed and spontaneously dying members of the population revealed a low rate of neoplasia and a variety of degenerative lesions that increased with age. These observations of a varied pathology and a low frequency of neoplasia provide justification for the continued development of the male LVG Syrian hamster as an animal model system for use in studies on the mechanism of both in vivo and in vitro aging.

High-frequency 1H NMR studies of the effects of growth factors and phorbol esters on normal Syrian hamster diploid fibroblast cells.

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T1), and spin-spin (T2) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (less than or equal to 100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T1 values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T2 values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986), Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T1 and T2 were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T2 values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-alpha-phorbol-12,13-didecanoate (4 alpha PDD). Treatment with either growth factor resulted in smaller T2 values, but a statistically significant decrease was not observed for PDD or 4 alpha PDD. The observed reductions in T2 values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.

NMR relaxation study of water protons in Syrian hamster fetal cells at 300 MHz.

The T1 and T2 relaxation times of water protons in two cell types in culture derived from Syrian hamster fetuses (normal primary or secondary fetal cells vs BP6T tumor cells derived from the normal cells transformed by carcinogens) were measured at 7.05 Tesla magnetic field (proton frequency = 300 MHz). The T1/T2 ratios and the correlation time, tau c, calculated from the T1/T2 ratio of cellular water protons, are significantly different in these two fibroblastic cell types of the same biological origin and with similar morphologies and growth rates in culture.

In vitro senescence of Syrian hamster mesenchymal cells of fetal to aged adult origin. Inverse relationship between in vivo donor age and in vitro proliferative capacity.

Normal diploid Syrian hamster dermal mesenchymal cell strains, regardless of the age of the tissue of origin, exhibit in vitro cellular senescence. The frequency of spontaneous escape from senescence and conversion to a permanent cell line is less than 5% among replicate flasks. The overall pattern of senescence of cells of fetal, neonatal, young adult (6 months) and aged adult (24 months) origin is similar in terms of the morphological changes and proliferative changes indicated by the reduction of saturation density, cloning efficiency and [3H]thymidine labeling index and by the increase in population doubling time and cell volume. However, the average maximum cumulative population doubling level is characteristic for each cell type: 13-day gestation fetal cells, 28.6; neonatal cells, 18.7; young adult cells, 13.8; aged adult cells, 11.1. Thus, the in vitro proliferative capacity of Syrian hamster mesenchymal cells is inversely related to the in vivo age of the donor.