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14 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS2018

Mechanoregulation of Proliferation, Differentiation, Senescence and Survival of Bone Marrow Primary Osteoprecursor Cells

Cell and animal studies conducted onboard the International Space Station and during the Shuttle program have provided extensive data illustrating bone degenerative responses to mechanical unloading in microgravity. Specifically CDKN1a/p21, an inhibitory modulator of cell cycle progression, is upregulated in osteoprecursor cells of the femur during 15-day spaceflight, suggesting that microgravity can block stem cell-based tissue regenerative process at the level of progenitor proliferation and differentiation. To study a potential role for CDKN1a/p21 in regulating osteogenic mechanosensitivity, we cultured primary bone marrow osteoprogenitor cells from CDKN1a/p21-null (p21-null) and wildtype mice with and without mechanical stimulation, and compared their morphological, proliferative, and in-vitro mineralization responses. Structural cell alterations due to mechanical stimulation were assessed by florescence labeling of f-actin cytoskeleton and focal adhesions. Mechanical stimulation of p21-null cells resulted in more pronounced cytoskeletal alignment with the axis of stretch than for wildtype cells. In addition, p21-null cells subjected to stretch loading also formed significantly more focal adhesions than wildtype cells. Combined these findings suggest that p21-null cells are structurally more responsive to stretch stimulation than the wildtype cells. Because osteoprogenitor cells are well known to respond to mechanical stimulation with increased proliferation, we also tested this response in p21-null cells. Results from those experiments show the proliferative capacity of mechanically stimulated p21-null cells far exceeded that of wildtype controls. Specifically, cell counts from 14, and 21 days post mechanical stimulation, show that p21- null cells to have a 4-fold increase in proliferation compared to wildtype. When the p21-null cell differentiation response to mechanical stimulation was evaluated, the p21-null cultuers elicited more extensive mineralization at earlier assessed timepoints than control cultures. Specifically, Von Kossa staining for mineralized matrix showed that the p21-null cells produced more than twice the mineralized surface area of wildtype cells, and at an earlier 7-day time point in culture. Taken together these results suggest that CDKN1a/p21 normally plays a role in negatively regulating osteoprogenitor proliferation and differentiation responses to mechanostimulation in bone. Findings of CDKN1a/p21's increased expression during spaceflight in microgravity also suggest not only a potential molecular mechanism for arresting regenerative bone growth in space, but potentially also a reduced impact for bone-formation-promoting exercise mechanostimulation. The findings described here constitute a novel role for p21 as a regulator of tissue regeneration in response to mechanical load stimulation, and also suggest a new promising molecular target to promote regenerative health in disuse conditions.

gravity↗
NASA NTRS1999

Cyclin E-p27 opposition and regulation of the G1 phase of the cell cycle in the murine neocortical PVE: a quantitative analysis of mRNA in situ hybridization

We have analyzed the expression patterns of mRNAs of five cell cycle related proteins in the ventricular zone of the neocortical cerebral wall over the course of the neuronogenetic interval in the mouse. One set of mRNAs (cyclin E and p21) are initially expressed at high levels but expression then falls to a low asymptote. A second set (p27, cyclin B and cdk2) are initially expressed at low levels but ascend to peak levels only to decline again. These patterns divide the overall neuronogenetic interval into three phases. In phase 1 cyclin E and p21 levels of mRNA expression are high, while those of mRNAs of p27, cdk2 and cyclin B are low. In this phase the fraction of cells leaving the cycle after each mitosis, Q, is low and the duration of the G1 phase, TG1, is short. In phase 2 levels of expression of cyclin E and p21 fall to asymptote while levels of expression of mRNA of the other three proteins reach their peaks. Q increases to approach 0.5 and TG1 increases even more rapidly to approach its maximum length. In phase 3 levels of expression of cyclin E and p21 mRNAs remain low and those of the mRNAs of the other three proteins fall. TG1 becomes maximum and Q rapidly increases to 1.0. The character of these phases can be understood in part as consequences of the reciprocal regulatory influence of p27 and cyclin E and of the rate limiting functions of p27 at the restriction point and of cyclin E at the G1 to S transition.

NASA Discipline Cell Biology↗
NASA NTRS2017

The Role of CDKN1a/p21 in Cellular Senescence of Bone Marrow Stem Cells Under Spaceflight Stressors

Spaceflight environments and their associated conditions, such as microgravity and space radiation, cause many biological functions formerly considered to be standard to behave in nonstandard ways. Exposure to microgravity has shown to induce deleterious effects in stem cell-based tissue regeneration, leading to immune system and healing response impairments as well as muscle and bone density loss. Such risks must be mitigated in order for long-term human space exploration to proceed. Thus, our work seeks to explore mechanisms of stem cell-based tissue regeneration that experience changes in spaceflight environments. Cellular senescence is a process of inducing cell cycle arrest that can be initiated by various stimuli. This function is influenced by two major pathways, controlled by p53 and pRB tumor suppressor proteins. p53 activity targets the cyclin-dependent kinase inhibitor gene p21Cdkn1a in osteogenic cell cycle arrest. Under conditions of mechanical unloading, stem cell-based tissue regeneration has shown to be decreased in both proliferation and differentiation, as many cells are arrested in progenitor states. p21 has shown upregulation in expression under conditions of microgravity, suggesting its role in regenerative bone formation arrest in space. p21 levels are found to be elevated independent of p53, suggesting a decrease in proliferation and regeneration without apoptosis, but rather through cell cycle arrest alone. Thus, we hypothesize that p21 is a mediator of cellular senescence in bone marrow stem cells. Culturing of bone marrow stem cells from wild type and p21 knockout mice under osteoblastogenic conditions will be completed to explore the role of p21Cdkn1a in stem cell proliferation and maturation. We believe that decreases in somatic stem cell differentiation may occur after spaceflight due to signal pathway alterations that result in downstream inhibition of genes involved in differentiation, preventing tissue from repairing and regenerating normally.

Stem Cells↗
NASA NTRS2023

Dual Genes of Muscle Cell Senescence: Unraveling the Radiation-Induced Roles of HDAC4 and MORC2a

As the human race ventures further into space and plans missions to explore the Moon and the expanse that lies ahead, addressing health challenges that may hinder exploration becomes an imperative task. However, limited research exists on the molecular mechanisms within skeletal muscle cells in response to long-term radiation-induced oxidative stress. Upon analyzing GLDS-426 from the GeneLab Repository, upregulation of the DNA damage response gene HDAC4 in space radiation conditions with 1G was observed, where radiation-induced oxidative stress increased DNA damage. DNA damage response gene MORC2a was found to be downregulated in space radiation conditions in 1G with a culture treatment of nanoceria, an antioxidant that alleviates the effects of oxidative stress and causes less DNA damage. Literature review further indicated that MORC2a recruits HDAC4 at the promoter to prevent the transcription of p21. Upregulation of p21 has been linked to cell senescence and muscle atrophy on Earth, but no research has studied its changes in radiation conditions. To address the knowledge gap regarding the HDAC4/MORC2a pathway impact on p21 expression in radiation, our experiment aims to silence HDAC4 and MORC2a in the Mus musculus C2C12 cell culture line. These cultures will be exposed to radiation to assess the effects on cellular senescence (Aim 1). Additionally, we will silence p21 and subject the cell cultures to radiation to measure muscle cell proliferation (Aim 2). Muscle atrophy in future long-term space missions can contribute to the risk of degenerative diseases and hinder the completion of tasks in space. Furthermore, exploring the impact of these genes on muscle atrophy may provide therapeutic insights for individuals experiencing skeletal muscle degeneration and elderly individuals affected by sarcopenia.

GL4HS↗
NASA NTRS1999

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

NASA Discipline Radiation Health↗
NASA NTRS1995

The tonotopic map in the embryonic chicken cochlea

The purpose of the present study was to determine the tonotopic map in the chicken cochlea at 19 days of incubation (E19) by obtaining characteristic frequencies (CFs) for primary afferents, labeling the characterized neurons, and documenting their projections to the papilla. The lowest and highest CFs recorded were 188 and 1623 Hz respectively. The embryonic tonotopic map coincided with maps reported for post-hatch chicks. There were no evidence that neurons selective to low frequencies project inappropriately to more basal locations of the embryonic papilla. Linear regression was used to estimate the frequency gradient (b = 0.037 +/- 0.012 In Hz/% [b +/- SEb]) and intercept (In C, where C = 111 Hz) of the semilog plot of frequency versus cochlear position (in % distance from apex). From these estimates the octave distribution was calculated to be 18.7%/octave or 0.58 mm/octave. These quantities were not significantly different from those found in post hatch chickens. We conclude that the tonotopic map of the avian cochlea for CFs between 100 and 1700 Hz is stable and relatively mature from age E19 to post-hatch day 21 (P21). The most striking sign of immaturity in the E19 embryo is the limited range of high CFs. We offer the hypothesis that, between the ages of E19 and P21, improvements in middle ear admittance alone or in combination with functional maturation of the cochlear base may be the principal factors responsible for the appearance of adult-like high CF limits and not an apically shifting tonotopic map.

NASA Discipline Neuroscience↗
NASA NTRS2002

Alteration of gene expression profiles in skeletal muscle of rats exposed to microgravity during a spaceflight

To clarify the mechanism of skeletal muscle wasting during spaceflights, we investigated whether intramuscular gene expression profiles are affected, by using DNA microarray methods. Male rats sent on the 17-day NASA STS-90 Neurolab spaceflight were sacrificed 24 hours after return to earth (MG group). Ground control rats were maintained for 17 days in flight-simulated cages (CS group). Spaceflight induced a 19% and 23% loss of tibialis anterior and gastrocnemius muscle mass, respectively, as compared to ground controls. Muscle RNA was analyzed by the Clontech Atlas DNA expression array in four rats, with two MG/ CS pairs for the tibialis anterior, and one pair for the gastrocnemius. Alterations in gene expression were verified for selected genes by reverse-transcription PCR. In both muscles of MG rats, mRNAs for 12 genes were up-regulated by over 2-fold, and 38 were down-regulated compared to controls. There was inhibition of genes for cell proliferation and growth factor cascades, including cell cycle genes and signal transduction proteins, such as p21 Cip1, retinoblastoma (Rb), cyclins G1/S, -E and -D3, MAP kinase 3, MAD3, and ras related protein RAB2. These data indicate that following exposure to microgravity, there is downregulation of genes involved in regulation of muscle satellite cell replication.

NASA Program Fundamental Space Biology↗
NASA NTRS2001

Effects of hypergravity exposure on the developing central nervous system: possible involvement of thyroid hormone

The present study examined the effects of hypergravity exposure on the developing brain and specifically explored the possibility that these effects are mediated by altered thyroid status. Thirty-four timed-pregnant Sprague-Dawley rats were exposed to continuous centrifugation at 1.5 G (HG) from gestational Day 11 until one of three key developmental points: postnatal Day (P) 6, P15, or P21 (10 pups/dam: 5 males/5 females). During the 32-day centrifugation, stationary controls (SC, n = 25 dams) were housed in the same room as HG animals. Neonatal body, forebrain, and cerebellum mass and neonatal and maternal thyroid status were assessed at each time point. The body mass of centrifuged neonates was comparatively lower at each time point. The mass of the forebrain and the mass of the cerebellum were maximally reduced in hypergravity-exposed neonates at P6 by 15.9% and 25.6%, respectively. Analysis of neonatal plasma suggested a transient hypothyroid status, as indicated by increased thyroid stimulating hormone (TSH) level (38.6%) at P6, while maternal plasma TSH levels were maximally elevated at P15 (38.9%). Neither neonatal nor maternal plasma TH levels were altered, suggesting a moderate hypothyroid condition. Thus, continuous exposure of the developing rats to hypergravity during the embryonic and neonatal periods has a highly significant effect on the developing forebrain and cerebellum and neonatal thyroid status (P < 0.05, Bonferroni corrected). These data are consistent with the hypothesized role of the thyroid hormone in mediating the effect of hypergravity in the developing central nervous system and begin to define the role of TH in the overall response of the developing organism to altered gravity.

Non-NASA Center↗
NASA NTRS2008

Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

Zhang, Ye↗
NASA NTRS2008

Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

Zhang, Ye↗
NASA NTRS2009

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems.

Zhang, Ye↗
NASA NTRS2017

Dose- and Ion-Dependent Effects in the Oxidative Stress Response to Space-Like Radiation Exposure in the Skeletal System

Exposure to space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized exposure to ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-week old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 mega electron volts per nucleon) or high-LET (sup 56) Fe ions (600 mega electron volts per nucleon) using either low (5 or 10 centigrays) or high (50 or 200 centigrays) doses at NASAs Space Radiation Lab at Brookhaven National Lab (NSRL/BNL). Tissues were harvested 5 weeks or 1 year after irradiation and bones were analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed for select groups by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) during the proliferative phase or the mineralizing phase, and differentiation was analyzed by imaging mineralized nodules (percentage surface area). Representative genes were selected for expression analyses, including cell proliferation (PCNA, Cdk2, p21, p53), differentiation (Runx2, Alpl, Bglap), oxidative metabolism (Catalase, GPX, MnSOD, CuZnSOD, iNos, Foxo1), DNA-damage repair (Gadd45), or apoptosis (Caspase 3). As expected, a high dose (200 centigrays), but not low doses, of either (sup 56) Fe or protons caused a loss of cancellous bone volume per total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; (sup 56) Fe (200 centigrays) inhibited median nodule area by more than 90 percent at 5 weeks and 1 year post-irradiation, compared to controls. At 5 weeks post exposure, irradiation with protons or (sup 56) Fe caused few changes in gene expression levels during osteoblastogenesis, although a high dose of (sup 56) Fe (200 centigrays) increased levels of Catalase and Gadd45. In addition, supplementing cell culture media with SOD protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET ((sup 137) Cs gamma) if irradiated in vitro, but had limited protective effects on high-LET (sup 56) Fe-exposed cells. In sum, exposure of mice to either protons or (sup 56) Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET (sup 56) Fe increased expression of redox-related genes and inhibited osteoblastogenesis, albeit to a limited extent. We conclude that high-LET irradiation impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss.

ionizing radiation↗
NASA NTRS2017

The Effects of CDKN1a/p21 on Oxidative Stress and Mitochondrial Function During Long Duration Spaceflight

Broad tissue degeneration and the failure of normal tissue regenerative processes in microgravity because of mechanical unloading are increasing concerns for sustaining life in space as the duration of future flight missions increases. Work in our laboratory has identified normal adult stem cell-based tissue regenerative processes, such as the formation of new bone, cartilage, and immune cells, as being particularly sensitive to the stresses of mechanical unloading in microgravity. Our studies have also identified the inhibition of differentiation of marrow mesenchymal stem cells and activation of CDKN1ap21-mediated cell cycle arrest in proliferative osteoprecursor cells on the bone surface as potential mechanisms for spaceflight-induced skeletal changes. This finding, in combination with the role of CDKN1ap21 as a suppressor of mammalian tissue regeneration, suggests that this gene could be responsible for suppressing stem cell-based tissue regeneration in response to disuse. In this work, we hypothesized that CDKN1ap21 regulates regenerative bone formation in response to alterations in mechanical load and tested this hypothesis by studying the skeletal phenotype and stem cell regenerative ability of juvenile (4-11 weeks old) and adult (18 weeks-12 months old) p21 (--) knockout (KO) mice. Additionally, we analyzed bone micro-architectural properties, bone formation rates and differentiation capacity of bone marrow stem cells (BMSCs) from male and female KO mice exposed to hindlimb unloading (HU) for 15-30 days. We found that juvenile KO mice exhibited increased femoral trabecular and cortical bone formation, whilst three-point bending of the tibias from KO mice showed decreased bone stiffness. Conversely, adult KO mice exhibited no significant differences in micro-architectural properties compared to WT (wild-type) but woven bone structure was indicative of rapid bone remodeling. Furthermore, cortical bone properties showed similar characteristics to aged bone, including increased cross-sectional area and perimeter, whilst three-point bending showed increased stiffness and toughness. Interestingly, in-vitro, KO mice exhibited increased differentiation and mineralized nodule formation in osteoblastogenesis assays compared to WT. Preliminary results from CDKN1ap21 KO mice subjected to HU suggest altered sensitivity to mechanical unloading resulting in decreased cortical thickness compared to WT mice. However, KO mice subjected to short and long-duration HU show increased in-vitro differentiation potential of BMSCs to from form mature, mineral-forming osteoblasts, indicating maintenance of regenerative potential. Analysis of bone formation rates, cell proliferation rates and key genes of interest are currently underway. These results indicate a novel role for CDKN1ap21 in load-dependent osteoprogenitor proliferation and differentiation and that deletion of CDKN1ap21 results in an age-dependent release of osteoblast proliferation inhibition and increased bone formation and turnover.

Stem Cells↗
NASA NTRS1992

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS).

Non-NASA Center↗