Identification of p53 unbound to T-antigen in human cells transformed by simian virus 40 T-antigen.

In several clones of SV40-transformed human cells, we investigated the relative amounts of large T-Antigen (T-Ag) and p53 proteins, both unbound and associated within complexes, with the goal of identifying changes associated with transformation and immortalization. Cells were transformed by wild type (wt) T-Ag, a functionally temperature sensitive T-Ag (tsA58) and other T-Ag variants. Western analysis showed that while most of the T-Ag was ultimately bound by p53, most of the p53 remained unbound to T-Ag. Unbound p53 remained in the supernatant after a T-Ag immunoprecipitation and p53 was present in two to fourfold excess of T-Ag. In one transformant there was five to tenfold more p53 than T-Ag. p53 was present in transformants in amounts at least 200-fold greater than in untransformed human cells. In wt and variant T-Ag transformants, including those generated with tsA58 T-Ag, large amounts of unbound p53 were present in both pre-crisis and immortal cells and when the cells were grown at permissive or non-permissive temperatures. We also found that in transformants produced by tsA58, an SV40/JCV chimeric T-Ag and other variants, T-Ag appeared to form a complex with p53 slowly perhaps because one or both proteins matured slowly. The presence in transformed human cells of large amounts of unbound p53 and in excess of T-Ag suggests that sequestration of p53 by T-Ag, resulting from complex formation, is required neither for morphological transformation nor immortalization of human cells. Rather, these results support the proposal that high levels of p53, the T-Ag/p53 complexes, or other biochemical event(s), lead to transformation and immortalization of human cells by T-Ag.

Immortalization of human cells by mutant and chimeric primate polyomavirus T-antigen genes.

Human fibroblasts were morphologically transformed with wild type and mutant SV40 T-antigens (T-Ags) and with SV40/JCV and SV40/BKV chimeric T-Ags. The transformants were then assayed for the attainment of immortal cell growth. Several observations relating T-Ag and T-Ag domains to immortalization were made. Approximately 10% of SV40-transformants became immortal. Transformants generated by transfection or infection of cells with C-terminal T-Ag deletion mutants of SV40 did not immortalize. SV40/JCV and SV40/BKV chimeric T-Ags, containing C-terminal sequences from JCV or BKV, immortalized cells more efficiently than did the intact SV40 T-Ag, suggesting that the C-termini of the JCV and BKV T-Ags contain an enhanced immortalization function. However, chimeras in which the N-terminal or proximal-central portions of T-Ag were composed of JCV sequences failed to immortalize but did induce transformation. Constructs in which the JCV T-Ag Rb binding domain was replaced with SV40 sequences transformed human cells, but again the cells failed to immortalize. Transformants and immortalized cell lines produced by some SV40/JCV chimeras, contained p53 which was unbound by T-Ag. This occurred under conditions where p53 from SV40 and SV40/BKV transformants was bound to T-Ag. This may reflect the reduced stability of the SV40/JCV T-Ags.

Glycosaminoglycan synthesis by human keratinocytes: cell growth and medium calcium effects.

The influences of cell density, differentiation, and medium calcium levels on glycosaminoglycan biosynthesis were evaluated in cultured human epidermal keratinocytes. Following metabolic labeling with [35S]-sulfate and [3H]-glucosamine under steady state conditions in "high" medium calcium (greater than 1.0 mMol), the majority of sulfated glycosaminoglycans remained associated with the cell layers, whereas hyaluronic acid, which was present in smaller amounts than the sulfated products, was about equally distributed between the medium and the cell layers. Of the sulfated glycosaminoglycans, heparan sulfate and chondroitin 4/6-sulfate were the major species and were present in roughly comparable amounts, whereas dermatan sulfate was quantitatively the lesser of the products. The effects of "low" medium calcium (0.3 and 0.025 mM) were complex, although a consistent decrease in the incorporation of the [3H]-glucosamine precursor was found at high cell density, probably reflecting a decrease in its intracellular specific activity. In "high" calcium cultures, there was a strong inverse correlation (r = -0.92) between keratinocyte cell number and cellular production of sulfated glycosaminoglycans, whereas no such relationship was evident in cultures grown in "low" calcium medium at comparable cell density. Because keratinocyte differentiation is inhibited in the low calcium conditions, the results suggest that the decrease in production of sulfated glycosaminoglycans by confluent keratinocytes may actually correlate with differentiation rather than with cell number.

Glycosaminoglycan synthesis by proliferating and differentiated human keratinocytes in culture.

Glycosaminoglycan (GAG) synthesis and compartmentalization were studied in populations of human neonatal keratinocytes under conditions of proliferation and terminal differentiation in vitro. Following isotopic labeling with the precursors [3H]glucosamine and [35S]sulfate, GAGs were extracted from the keratinocytes into several operationally created compartments associated with the cells and extracellular matrix. Chondroitin sulfate and heparan sulfate accounted for the majority of the incorporated label in all preparations. Although total sulfated GAGs per culture increased from proliferative to differentiated conditions, GAG content normalized to the DNA content of the cultures demonstrated the reverse trend. This was particularly evident for the chondroitin sulfates, which declined 60-70% in the differentiated cultures. Furthermore, label incorporation into chondroitin and heparan sulfates revealed a relative compartmental shift to a trypsin-accessible site upon keratinocyte differentiation. An analysis of heparan sulfate structure by characterization of the oligosaccharide products resulting from low pH nitrous acid deaminative degradation provided evidence that the parent material from differentiated keratinocytes contains a larger region of N-sulfated glucosamine residues unassociated with ester sulfate groups. The correlation of variations in GAG content and compartmentalization with the growth condition of human keratinocytes constitutes evidence that this heterogeneous group of cell surface-associated carbohydrates is involved in some aspect of cell function associated with growth control or differentiation. Furthermore, the apparent differences in heparan sulfate primary structure indicate that there is structure-function specificity to this association.

The distinctive pattern of proteoglycan and glycosaminoglycan free chain synthesis by cultured human epidermal keratinocytes.

The in vitro synthesis of proteoglycans and glycosaminoglycan free chains was studied in human epidermal keratinocytes. Preconfluent and confluent cultures established on 3T3 feeders were steady state labeled with [35S]-sulfate and [3H]-glucosamine after removal of the 3T3 cells. Products in nonionic detergent extracts of keratinocytes and in the medium were analyzed in the presence of protease inhibitors. Glycosaminoglycans as proteoglycans and as free chains were defined by susceptibility or resistance, respectively, to alkaline borohydride reduction. Products associated with the cells were approximately 30% proteoglycans and approximately 70% glycosaminoglycan free chains, whereas in the medium virtually all was proteoglycan. The heparan and chondroitin sulfate proteoglycans were small compared to those of many other cell types. Their Kav on Sepharose CL-4B was 0.56 (estimated 50 kDa), whereas the free chain Kav was 0.74 (estimated 12 kDa). Relative amounts of the sulfated products varied with confluence and differentiation; heparan and chondroitin sulfates were equally represented within the free chains and proteoglycans of the cells in preconfluent, proliferating cultures, whereas in postconfluent, differentiated cultures the major labeling was in the heparan sulfate products, consistent with our prior reports (J Invest Dermatol 88:215-9, 1987 and 91:492-8, 1988). The cellular localization of the products was probed with glycosaminoglycan degrading enzymes added to isotopically prelabeled cultures. The proteoglycans appeared to be located on the external surface of plasma membranes, whereas the glycosaminoglycan free chains resisted digestion and are either intracellular or membrane associated, but otherwise inaccessible. These data establish the distinctive pattern of low Mr proteoglycans and abundant cell-associated glycosaminoglycan free chains synthesized by keratinocytes.

Persistence of the SV40 early region without expression in permissive simian cells.

SV40 containing recombinant vectors were introduced into permissive simian, non-permissive rodent and semi-permissive human cell lines, and assayed for transformation. All mouse and human cell clones expressed T-antigen (T-Ag) and were morphologically transformed when they contained only the wt T-Ag gene (E-SV40) or the entire wt viral genome with an interrupted late region. However, of 63 simian clones with these recombinant vectors, none became morphologically transformed and T-Ag containing cells were rare or absent. Nearly all simian cell lines made either no detectable early SV40 RNA or only small amounts of viral RNA but contained viral DNA restriction fragments similar to those in the original recombinant vectors. Functional T-Ag genes were recoverable from several cell clones and used to regenerate infectious virus. Hence, T-Ag gene expression had been suppressed. We found two conditions where T-Ag expression was activated. In a BSC-1 cell line containing E-SV40 DNA, subsequent introduction of a vector with a functional viral late coding region (L-SV40) resulted in the appearance of T-Ag and transformation. These findings suggest that L-SV40 sequences activate or enhance T-Ag expression and that this activation requires a functional Vpl gene. We found also, that vectors with E-SV40 DNA from the bipartite variant EL-SV40 consistently transformed simian CV-1 cells. Transformation was shown to be effected by the multiple alterations present in the regulatory region of this variant.

Papillomavirus infection of aged Persian cats.

Papillomavirus infection was confirmed in 2 Persian cats with sessile hyperkeratotic skin lesions. Skin lesions were not typical papillomas as found in other species. Papillomavirus were demonstrated in negative stain preparations of homogenized tissue and within nuclei of cells in the stratum granulosum. Papillomavirus group-specific antigens were detected within nuclei corresponding to those containing virions. Attempts to transmit this disease to other cats or propagate the virus in tissue cultures were unsuccessful. A 7.8-kilobase DNA molecule was present in low-stringency Southern blots using a bovine papillomavirus type 1 cloned DNA probe. In reverse Southern blots, the cat papillomavirus hybridized under conditions of low stringency with all papillomavirus genomes tested. Combined with limited restriction endonuclease restriction mapping, the above information indicates that the feline cutaneous papillomavirus is a unique virus type and thus expands the list of hosts known to be infected by papillomaviruses.

Feline hemobartonellosis.

H. felis is a rickettsial parasite that causes hemolysis and sequestration of feline erythrocytes. It should be considered as a potential primary pathogen or opportunist in any cat presented with signs ranging from episodic malaise to acute anemic collapse. Diagnosis requires visualization of the organism in properly prepared blood smears. Treatment uses antirickettsial drugs, corticosteroids, and supportive measures. Clinical recovery requires immune containment of the organism. Treatment does not eliminate the organism from the host. Carrier cats may relapse when their immunity is severely compromised by other diseases such as FeLV. Transmission is presumed to be by blood-sucking parasites and possibly bite wounds between cats. Prevention requires prudent health management of cats. Future advances in the knowledge of the disease will relate mainly to the development of a diagnostic technique that will allow identification of all infected cats.

Hearing loss due to combined effects of noise and sodium salicylate.

This study reports on hearing loss due to the combined effects of noise and sodium salicylate. A group of 10 adult chinchillas was used to test the hypothesis that the combined effects of prolonged exposure to noise and continuous salicylate intoxication will result in a larger Temporary Threshold Shift (TTS) in the audibility curve than either of the agents alone can produce. Extensive behavioral training was used to determine hearing thresholds. Though use of shock avoidance techniques, the animals were trained to respond to tones by jumping a low barrier. After demonstrating sufficient ability in behavioral training, the animals were surgically rendered monaural. After recovery from surgery, monaural threshold data were obtained. The animals were exposed in a chamber to 85 dbA broadband noise for 48 h to measure TTS. The chinchillas were allowed to recover for a minimum of 45 days; then a second monaural audibility curve was obtained. The chinchillas were then injected subcutaneously with sodium salicylate at 6 h intervals to establish TTS-producing serum salicylate levels. After 36 h of salicylate exposure, the animals were placed in a noise chamber. For the next 42 h the animals were exposed to both sodium salicylate and 85 dbA noise. The results of this study indicated the chinchillas' hearing threshold is reduced in sensitivity by approximately 35 dB due to broad-band noise exposure of 85 dBA intensity. Serum salicylate levels of 20-40 mg per 100 ml resulted in a TTS of 30 dB on the average. The combination of noise and sodium salicylate exposure produced a temporary hearing loss of approximately 55 dB. These data suggested that the combination of prolonged noise and salicylate exposure may result in a larger hearing loss than either agent alone can produce.

Field tests of interspecific resource-based competition among phytoplankton.

The hypothesis that interspecific resource-based competition caused the spring and summer vertical segregation of phytoplankton species was tested in Lake Tahoe (California/Nevada). Two species (Cyclotella glomerata Bachmann and Synedra radians Kütz.) became dominant at different depth intervals (0-30 m and 60-90 m, respectively). Experimental transplants of assemblages between depths demonstrated asymmetrical competition. In the phosphate-limited region near the surface, growth of S. radians declined in the presence of C. glomerata. However, growth of C. glomerata was not affected by the presence of S. radians in the light-limited region at depth. This study provides field verification of resource-based competition theory.

Presence and role of glycosaminoglycans in amyloidosis.

Though the presence of glycosaminoglycans in amyloid deposits has been recognized for a long time their role in the pathogenesis of the disorder has remained elusive. As shown here, liver and spleen of human patients with secondary amyloidosis contain 5 to 10 times the amount of glycosaminoglycans as normal organs. Of the three major glycosaminoglycans measured, the heparan sulfate fraction showed the largest increase. In mice where amyloidosis was induced by the injection of casein and enhancing factor (accelerated model) 35SO4-labeled, or Alcian blue stained glycosaminoglycans appeared as early as and at the same location as proteins detected by Congo red staining which was about 2 days after initiation of the procedure. When glycosaminoglycan synthesis was followed in liver and spleen slices of control and experimental animals a significant increase in rate was found in the spleen of the experimental mice. Though there was an increase in heparan sulfate synthesis the major contribution to the overall increase was made by the chondroitin sulfates in the accelerated as well as in the standard induction model. In addition, unlike in the human disorder the chondroitin sulfates were the major glycosaminoglycans which had accumulated in the spleens of animals which had amyloidosis induced by the long term standard procedure (6 weeks) as measured by isolation and uronic acid analysis. The data presented here show that glycosaminoglycans appear to play an important and perhaps direct role in the process of amyloid deposition in the human disease as well as in the experimentally induced disorder in animals.

Growth-related variations in the glycosaminoglycan synthesis of ultraviolet light-induced murine cutaneous fibrosarcoma cells.

Glycosaminoglycan synthesis was studied in cell populations of ultraviolet light-induced murine cutaneous fibrosarcoma cells under conditions of varying growth rates in vitro. After labeling with the precursors, 3H-glucosamine and 35SO4, sulfated glycosaminoglycans recoverable by direct proteolysis of the culture monolayers increased approximately 5-fold on a per cell basis from sparsely populated, exponential cell cultures (greater than 85% of cells in S, G2, or M phases) to stationary cultures inhibited by high cell density (greater than 50% of cells in G1). Within this cell surface-associated material, the relative ratio of heparan sulfate to the chondroitin sulfates was approximately 60/40% under conditions of exponential growth; in the growth-arrested cultures, the reverse ratio was found. The substratum attached material, obtained from the flask surface after ethyl glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-mediated detachment of the monolayers, contained relatively more hyaluronic acid, heparan sulfate, and chondroitin sulfates in the most actively proliferating cultures compared with the growth-inhibited cell populations. Furthermore, heparan sulfate and the chondroitin sulfates, which were enriched in the substratum material and in the cell pellet of exponential cultures, showed a relative shift to the cell surface-associated compartment (releasable by mild trypsinization after EGTA-mediated cell detachment) and to the compartment loosely associated with the pericellular matrix (i.e., released into the supernatant during detachment of the monolayers in the presence of EGTA). These results demonstrate that a variety of differences in the quantities, relative compositional ratios, and cell compartment distributions of hyaluronic acid and sulfated glycosaminoglycans occur in fibrosarcoma cell populations which vary in their rate of cell growth consequent to cell density in culture.

Host range analysis of simian virus 40, BK virus and chimaeric SV40/BKV: relative expression of large T-antigen and Vp1 in infected and transformed cells.

Simian virus 40 (SV40) persists in Rhesus monkeys and productively infects cultured simian kidney cells. In contrast to the closely related human virus BKV, SV40 is known to propagate inefficiently in human embryonic kidney (HEK) cells and human fibroblasts (HFF). We examined the growth of SV40, BKV and the chimaeric genome virus, SV40/RFV, in several types of human cells. We analysed replication, expression of T-Ag and Vp1 capsid proteins, and cytopathic effects (CPE). We also compared T-Ag and Vp1 expression in infected versus transformed HFF cells. Although SV40 DNA replicated in HFF and in one subtype of HEK cells, viral DNA accumulated slowly and did not reach high levels until six to eight weeks after transfection. In HFF or HEK cells there was little T-Ag produced but Vp1 was produced in significant amounts in HFF cells. In HFF cells the Vp1/T-Ag ratio was approximately 200: 1, and expression of the viral late region appeared to inhibit expression of the T-Ag gene. In contrast, BKV and the SV40/RFV hybrid propagated well in HEK and HFF cells. The Vp1/T-Ag ratios were also high in BKV and SV40/RFV infected HFF cells but more T-Ag was produced with BKV and SV40/RFV Because SV40/RFV contained the RFV capsid genes but a SV40 T-Ag gene and regulatory region, the human versus simian host range of SV40 was controlled by the viral late region, or one or more capsid proteins. This suggested that the production of small amounts of T-Ag could not by itself account for poor growth of SV40 in HFF cells and that very small, barely detectable amounts of T-Ag were sufficient to activate Vp1 gene expression. Also, although some feature of the SV40 late region prevented rapid growth of the virus in HFF cells, poor virus growth could not be explained by the inability to produce a significant amount of Vp1. Although little T-Ag accumulated in SV40 infected HFF and HEK cells, transformants contained large amounts of T-Ag. In transformants there was a reversal of the Vp1/T-Ag ratio, such that T-Ag was now in 10-20 fold greater amount than Vp1. The relatively large amount of T-Ag in transformants could be accounted for by the relative absence of Vp1, which may inhibit T-Ag production, or by integration of the T-Ag gene at a site in the cell DNA which allows for elevated T-Ag gene expression.

DNA sequences outside the simian virus 40 early region cause downregulation of T-antigen production in permissive simian cells.

Using a series of modified wtSV40 and early region SV40 DNAs we assayed the effect of viral late region sequences on T-antigen production by the SV40 early region. We found that SV40 late region (L-SV40) DNA sequences reduced T-antigen (T-Ag) production by the SV40 early region (E-SV40) when both viral regions were linked as they are in wtSV40 DNA. This was demonstrated by Western analysis which showed that E-SV40 DNA produced 10 times more T-Ag than wtSV40 DNA L-SV40, with its own promoter but unlinked to E-SV40 DNA, also greatly inhibited T-Ag production when it was contrasfected with E-SV40. Therefore, L-SV40 DNA inhibited T-Ag production by E-SV40 DNA when present in cis or in trans. We have shown that expression of the SV40 late transcription unit dominated that of the early (T-Ag gene) transcription unit because late region RNA accumulated to much higher levels than early viral RNA. However, in contrasfected cells L-SV40 DNA did not replicate to higher levels than E-SV40 DNA. We offer a model for control of T-Ag expression in which a relatively small amount of T-Ag activates late transcription at the expense of T-Ag gene transcription and that this represents a switch from early to late viral gene expression. We suggest that when activation of the late transcription unit occurs at the late promoter, expression of the T-Ag gene is greatly reduced. The L-SV40 promoter may inhibit T-Ag gene transcription by sequestering cellular factors required for early transcription, factors which may be present in limited amounts. We suggest further that activation of late transcription allows for the necessary production of large amounts of capsomeres and virions and downregulation of early transcription prevents the early region from interfering with capsid synthesis. We tested the model using a construct with a wild-type T-Ag gene but with mutations in the SV40 major late promoter which prevent the promoter from being bound by cellular repressors of late transcription. We found that this construct, which overproduces late SV40 RNA, was defective for T-Ag production. This indicates that activation of the late promoter results in repression of T-Ag gene expression.