Thomas J. King Jr. 1921-2000.
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Cloning, Organism↗
Biomedical subjects
R G McKinnell
Publications and source records attributed to R G McKinnell.
Genomic studies of the Lucké tumor herpesvirus (RaHV-1).
Ranid herpesvirus 1 (RaHV-1) is the etiological agent of the Lucké renal adenocarcinoma of the North American leopard frog Rana pipiens. Construction of cosmid libraries containing RaHV-1 DNA inserts allowed the derivation of a BamHI map for the viral genome. Summation of fragment sizes indicates that the genome is 217 kbp in size, a value in accordance with the most recent published estimate (220 kbp) obtained by field-inversion gel electrophoresis. The DNA sequence of the 39,757-bp insert in 1 cosmid (cos54) was determined and was predicted to contain 21 complete and 3 partial genes. In all, 12 genes have distant counterparts in a fish herpesvirus (ictalurid herpesvirus 1) and are present in 2 blocks, 1 of which is relatively inverted. This indicates that RaHV-1 belongs to the fish virus lineage of the herpesvirus family rather than to the lineage populated by mammalian and avian viruses. The remaining 12 genes in cos54 lack counterparts in any other herpesvirus. One of these encodes a putative DNA (cytosine-5) methyltransferase. This raises the possibility that biological processes induced in the host by RaHV-1 might involve methylation of cellular DNA by the viral enzyme.
Adenocarcinoma↗
Lucké renal adenocarcinoma, an anuran neoplasm: studies at the interface of pathology, virology, and differentiation competence.
The northern leopard frog, Rana pipiens, is vulnerable to a herpesvirus-induced renal tumor. The Lucké renal adenocarcinoma is metastatic as a function of temperature. The cloning procedure of nuclear transplantation has been used to study the differentiation potential of the tumor genome. This paper summarizes current studies of the pathology, virology, and differentiation competence of the Lucké tumor.
Adenocarcinoma↗
Pronephric carcinoma: chromosomes of cells rescued from apoptosis by an oncogenic herpesvirus detected with a polymerase chain reaction.
The amphibian pronephros is fated to die during early development. Pronephric cells undergo apoptosis and their function is replaced by the mesonephros, which becomes the functional kidney of the adult frog. Tadpoles of the northern leopard frog, Rana pipiens, were inoculated with a Lucké tumour herpesvirus (LTV) preparation. Most of the animals developed typical Lucké renal carcinomas at metamorphosis. Fewer developed carcinomas of the pronephric cell type. A pronephric carcinoma, rescued from apoptosis by the herpesvirus, was harvested from a post-metamorphic frog. The tumour was judged to be pronephric by its anatomical location (in the anterior part of the body) and because both mesonephric kidneys were intact and tumour-free upon removal of the tumour mass. A tumour fragment was fixed for histological examination, which confirmed that the tissue was a renal carcinoma. A further fragment was subjected to short-term culture in order to produce metaphase cells for cytogenetical analysis. Based upon silverstained nucleolar organizing region numbers, 14 of 15 metaphase cells were estimated to have the diploid number (2N = 26) of chromosomes and a karyotype was constructed which did not appear to differ from that of normal cells. A single cell was estimated to be tetraploid (4N = 52). This is the first report of chromosomes of a pronephric Lucké carcinoma. LTV replicates only in tumour tissue maintained in the cold. Because the frog in this study had been maintained in the laboratory at 22 degrees C for about 10 months, no viruses would have been detectable with electron microscopy. However, the presence of Lucké herpesvirus DNA was detected in tumour homogenates by polymerase chain reaction amplification of a 1.2 kbp Hind III restriction fragment of the LTV DNA. The presence of LTV DNA provided assurance that the rescued pronephric tumour was indeed a Lucké carcinoma.
Animals↗
Fate of herpesvirus DNA in embryos and tadpoles cloned from Lucké renal carcinoma nuclei.
The Lucké renal carcinoma of the northern leopard frog, Rana pipiens, has a herpesvirus aetiology. Lucké tumour nuclei inserted into enucleated frogs' eggs produce development to the swimming tadpole stage. Tissue from these tumour nuclear transplant animals can be induced to survive and differentiate further by allografting to normal tadpoles. We wished to ascertain whether the aetiological agent, the Lucké tumour herpesvirus (LTV), persists in the animals produced by tumour nuclear transplantation. The polymerase chain reaction was used to amplify a 1.2 kbp Hind III restriction fragment of LTV DNA in whole animal homogenates prepared from tumour nuclear transplant tadpoles and normal tadpoles fertilized in vitro. The LTV fragment was not present in the majority (31 of 34) of the cloned animals derived from tumour nuclei, nor was it present in any of five normal tadpoles. Either the 1.2 kbp fragment of LTV DNA was eliminated from most of the cloned animals during the massive reprogramming of the neoplastic genome initiated by insertion of the tumour nuclei into egg cytoplasm, or the nuclei selected for transplantation were primarily those lacking this fragment of LTV DNA. The limited development of the tumour nuclear tadpoles was probably not due to the presence of these viral sequences, but rather reflected the limited plasticity of the tumour cell genome as assayed by nuclear transplantation. Failure to detect the 1.2 kbp fragment of LTV DNA in the majority of mitotic progeny of the Lucké tumour genome does not imply that other parts of the viral genome do not persist.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The presence of DNA sequences of the Lucké herpesvirus in normal and neoplastic kidney tissue of Rana pipiens.
The Lucké tumour herpesvirus (LTV) is the aetiological agent of the Lucké renal adenocarcinoma of the northern leopard frog, Rana pipiens. LTV virions can be detected by electron microscopy in renal adenocarcinomata after prolonged exposure to low temperature. Tumours maintained at warm temperatures do not contain viral particles. To gain insight into the processes of viral infection, replication and oncogenesis, evidence was sought for the presence of LTV DNA in warm renal tumours and normal renal tissue. The polymerase chain reaction was used to amplify a Hind III restriction fragment of LTV DNA in tissue homogenates. LTV DNA was detected in a significant percentage of normal kidneys as well as in "virus-free" warm tumours and virus-containing cold tumours.
Adenocarcinoma↗
Cytogenetic analysis of triploid renal carcinoma in Rana pipiens.
To provide a cytogenetic marker for nuclear transplantation experiments, triploid Rana pipiens embryos were produced. These embryos were injected with Lucké tumor herpesvirus. The chromosome profile of a renal carcinoma that developed in one of these triploid embryos was compared to the chromosomal profiles of a naturally occurring diploid renal carcinoma and a diploid renal tumor maintained as serial anterior eye chamber allografts for over 7 yr. Examination of Ag-NOR-stained chromosome spreads from the putative triploid and naturally occurring putative diploid tumor revealed the expected results. The vast majority of the chromosome spreads, 54/57 (95%) and 6/7 (86%), respectively, displayed euploid chromosome and Ag-NOR profiles: 3N = 39 with three Ag-NORs at the secondary constrictions in the long arm of chromosome 10 (10q) and 2N = 26 with two Ag-NORs in 10q. Chromosome profiles from the long-term allografted tumor were highly aneuploid (82%) and, based on their Ag-NOR content, displayed variations in their 2N, 3N, and 4N numbers. These data indicate that the majority of recently transformed triploid Lucké tumor cells can provide donor nuclei suitable for the characterization of developmental potential.
Animals↗
Genomic plasticity of the Lucké renal carcinoma: a review.
The differentiation potential of the Lucké renal carcinoma of the northern leopard frog, Rana pipiens, can be characterized by the nuclear transplantation procedure. Transplantation of tumor nuclei into activated and enucleated ova results, in the best of cases, in swimming larvae which fail to feed. The larvae die in about 10 to 14 days. Rescue of tumor nuclear transplantation tadpole tissue, destined to die, has been accomplished by allografting fragments of that tissue to normal hosts. The allografts persist and differentiate a diversity of tissues which cannot be distinguished by histological analysis from allografted normal control tissue. Allografts are an imperfect mode of assay for histological competence because of the immune response of the host. Lymphocytes and eosinophils invade the grafts in about 40 days. The host immune response occurs in both experimental and control allografts. Consequently, we believe that added histogenetic potential exists in the genome of the Lucké renal carcinoma. We propose that unexpressed differentiative potential of the grafted tissue can be extracted by abrogation of the immune response of the host. A herpesvirus is the etiological agent of the Lucké renal carcinoma. We currently seek to detect viral DNA in tissue derived from tumor nuclear transplant embryos. The presence of the viral genetic material in normal mitotic progeny of Lucké tumor cells, if demonstrated, raises the question of the long-term stability of differentiated cells derived from a virus tumor. Alternatively, absence of viral DNA in the tumor nuclear transplant tissue would suggest that normal differentiation ensues after elimination of the oncogenic DNA from that tissue. Loss of viral DNA may prognosticate stable differentiation.
Animals↗
Allografts of tumor nuclear transplantation embryos: differentiation competence.
The developmental potential of nuclei can be studied by nuclear transplantation. Although amphibian blastula nuclei and other early embryonic nuclei are totipotent, to our knowledge no nucleus from an adult cell has ever been shown to be totipotent by this procedure. Transfer of Lucké renal carcinoma nuclei into enucleated eggs results in prefeeding swimming tadpoles. Inasmuch as these tadpoles die, rescue of this pluripotential tissue was attempted by grafting fragments of triploid tumor nuclear transplant tadpoles to the tails of normal diploid Rana pipiens hosts. Grafts of tumor nuclear transplant tadpole tissue were histologically indistinguishable from grafts of normally fertilized embryos and developed normal-appearing structures such as complete eyes, well-differentiated neural tissues, kidney tubules, and gut epithelium. Moreover, histological differentiation in tumor nuclear transplant grafts was comparable to that observed in 50-day-old normal larvae. Grafting enhanced the survival of tumor nuclear transplant tissue from no more than 14 days as part of the donor tadpole to 40 days at which time the grafts were harvested as healthy tissue. Thus, both differentiation and survival of tumor nuclear transplant tissue were augmented with the grafting procedure. Cytophotometric analysis of ploidy was used to confirm the tumor origin of the donor tissue.
Animals↗
Neoplastic cells. Modulation of the differentiated state.
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Animals↗
Vascular dissemination of tumor cells in relation to temperature-dependent metastasis in frogs.
Metastasis is a temperature-related phenomenon in the North American leopard frog (Rana pipiens) affected with the Lucké renal adenocarcinoma. The frog is a poikilothermic vertebrate whose internal body temperature closely follows that of the environment, and tumor-bearing frogs living in a warm (28 degrees C) environment have a much higher incidence of metastasis (greater than 75%) than those in cold (4 degrees C) conditions (less than 6%). In this investigation it was found that Lucké tumor cells labeled with fluorescein isothiocyanate could be detected in frozen sections of all organs examined within 15 minutes, regardless of whether the hosts were adapted to a warm or a cold environment. Separate experiments involving inoculation of isotope-labeled tumor cells demonstrated that large numbers of cells arrived in the liver and other organs of animals kept at either end of the temperature range. These findings show that the infrequency of metastasis by Lucké adenocarcinomas in chilled frogs is not due to failure of dissemination of tumor cells consequent upon temperature-mediated changes in blood viscosity or flow patterns. Thus they establish the important baseline that the effects of temperature on metastasis in this vertebrate are exerted directly on the tumor cells and/or the internal environment of the host and that the system therefore provides new opportunities for probing the biology and biochemistry of tumor metastasis.
Adenocarcinoma↗
Feeding tadpoles cloned from Rana erythrocyte nuclei.
Diploid frog nuclei from differentiated somatic cells, transplanted into enucleated eggs to determine whether cell specialization generally involves irreversible genetic changes, have shown that nuclei from specialized somatic cells still contain the genes specifying the cell types and organ systems of swimming tadpoles. However, those tadpoles failed to feed and did not survive beyond the initial tadpole stages. Here we report that, after incubation in oocytes, triploid erythrocyte nuclei from juvenile frogs of Rana pipiens directed the formation of feeding tadpoles that survived up to a month and had differentiated hind limb buds. These tadpoles occurred at a high yield and showed the most extensive development so far obtained from documented differentiated somatic nuclei.
Animals↗
The genome of frog erythrocytes displays centuplicate replications.
Seven nuclear lines derived from erythrocyte nuclei of Rana pipiens were produced by serial nuclear transplantation into oocytes and eggs. Even at the termination of the experiments, embryos and tadpoles developed in the eighth transplant generations. Thus, there was no evidence that the mitotic progeny of the erythrocyte nuclei lost their ability to replicate their genomes and continue cell cycling. We conclude that the genome of noncycling and terminally differentiated erythrocytes maintains its potential for widespread replication and extensive reversal of gene function in excess of a hundred (centuplicate) cell cycles.
Animals↗
Degradation of basement membrane collagens by metalloproteases released by human, murine and amphibian tumours.
In this investigation it has been found that naturally-occurring (i.e. indigenous, not transplanted) tumours of diverse organs in a spectrum of vertebrates from frogs to man can secrete enzymes which degrade basement membrane collagens (type IV and V). The enzymes are inhibited by chelating agents (EDTA) but not by other protease antagonists and are, therefore, specific metalloproteases. Individual tumours do not necessarily secrete collagenases active against all collagen types (I, IV and V) and release of these different enzymes does not, therefore, appear to be coordinated. These biochemical findings support those reported for serially transplanted tumour cell lines and provide a plausible mechanism for the destruction of basement membranes and stromal collagen fibres observed morphologically in tumour spread.
Adenocarcinoma↗
Temperature-dependent metastasis of the Lucke renal carcinoma and its significance for studies on mechanisms of metastasis.
Metastasis is temperature dependent in the renal adenocarcinoma of the North American leopard frog, Rana pipiens. Widespread, multiple, metastatic colonies occur in tumor-bearing frogs kept at 28 degrees C for 50 days while tumor-bearing frogs kept at 7 degrees C for 98 days or more have either no secondary deposits or they have only an occasional small metastatic nodule. An attractive aspect of the frog tumor is that invasion and metastasis can be permitted or inhibited by the manipulation of temperature alone-no exogenous chemicals or drugs are required for the effect. Because of this, biological variables which reproducibly and specifically associate with metastasis permissive conditions when ambient temperature is cycled between permissive and inhibitory values are strong candidates for being causal elements in the multistep process leading to metastasis. Intravascularly injected labelled renal tumor cells reached all organs studied in as little as 15 minutes at both metastasis restrictive and permissive temperature. The results with tumor cell inoculation dispose of the possibility that failure of metastasis in chilled animals is due to cold-induced changes in blood flow. Histologically typical metastatic colonies developed in frogs, kept at the permissive temperature, after injection with disaggregated tumor cells which were previously cryopreserved. Frog tumors elaborate type I collagenase in a temperature dependent manner. Type IV collagenase has been demonstrated as well. Tumor cell detachment in vitro, assembly and disassembly of tumor cell cytoplasmic microtubules, and invasion in vitro, are all temperature dependent.
Animals↗
Temperature-dependent dissociation of Lucké renal adenocarcinoma cells.
Fragments of Lucké renal adenocarcinoma were subjected to dissociation by rapid shaking after exposure to a divalent cation-free electrolyte solution, with or without 5 X 10(-4) M ethylenediaminetetraacetate (EDTA), at 7 degrees C and 28 degrees C. More cells detached at 28 degrees C than at 7 degrees C. Dissociation of cells from normal mesonephros fragments was minimal at both temperatures. It has been shown else-where that this frog tumor elaborates collagenase in a temperature-dependent manner. More collagenase is detected at 30 degrees C than at 7 degrees C. Normal kidney elaborates low levels of collagenase at both temperatures. Because our results suggested the possibility that some dissociation of the tumor cells may have been attributable to tumor-elaborated collagenase, we studied the effect of two collagenase inhibitors on dissociation. Both EDTA at high concentration and cysteine inhibit collagenase and both diminished tumor-cell dissociation.
Adenocarcinoma↗
Cytoplasmic microtubules of normal and tumor cells of the leopard frog. Temperature effects.
The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28 degrees C; similar results were obtained at 20 degrees C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7 degrees C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28 degrees C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 70 degrees C. However, while normal cells could effect a recovery at 7 degrees C after the removal of nocodazole, PONKT-4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28 degrees C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7 degrees C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.
Adenocarcinoma↗
Artificially induced metastasis by cells from spontaneous Lucké renal adenocarcinoma.
This communication reports the development of tumour colonies in various organs after vascular dissemination of disaggregated cells from spontaneously arising Lucké renal adenocarcinomata in Cyclosporin-A-treated allogeneic frogs, Rana pipiens. The sites of tumour cell colonies were mesonephros, lung, bladder, mesentery, fat body and muscle. It was also found that digestion of these tumours with collagenase is an effective means of obtaining sufficient dissociated viable cells for large experiments and that cryo-preservation does not abrogate the ability of these cells to form metastatic deposits. This report, therefore, introduces a new tumour system for the study of factors affecting metastasis using naturally occurring tumours.
Adenocarcinoma↗