Barid B. Mukherjee

Developmental Expression of Osteopontin (OPN) mRNA in Rat Tissues: Evidence for a Role for OPN in Bone Formation and Resorption

Osteopontin (OPN) is a 34-kDa, highly-phosphorylated glycoprotein with cell attachment properties that is a prominent constituent of the bone matrix. To aid in elucidating the function of this protein we have studied the cellular expression of OPN mRNA during the formation, growth and maturation of rat calvarial (membranous) and tibial (endochondral) bone. From Northern hybridization analysis OPN expression was demonstrated in the kidney and gravid uterus as well as in bone tissues. Compared to collagen, the expression of OPN was low in early bone formation but increased subsequently and reached peak levels in 14-day-old bone. However, both the collagen and OPN mRNAs decreased markedly thereafter and remained low in young adult bone. From in situ hybridization studies using a [35S]-labelled rat OPN cRNA probe, OPN mRNA was localized to osteoblastic cells in newly-forming calvariae, jaw bones, and in the metaphyseal and periosteal bone of the tibia. In contrast to bone sialoprotein (BSP), which is expressed almost exclusively by osteoblasts at sites of de novo bone formation, OPN transcripts were present in cells lining both endosteal and periosteal bone surfaces, and in osteocytes. Moreover, expression of OPN persisted during the subsequent growth and remodelling of both membranous and endochondral bone and was expressed at particularly high levels by bone cells and hypertrophic chondrocytes at sites of osteoclastic resorption. In the more mature bone of young adult rats OPN expression was significantly reduced but remained detectable in bone cells lining periosteal and endosteal surfaces and in the primary and secondary spongiosa of the tibia. These studies on the developmental expression of OPN support the concept of a multifunctional role for OPN in bone formation and remodelling. Thus, the expression of OPN by osteoblasts early in bone development is consistent with a role for this protein in the formation of bone matrix, whereas the peak expression of OPN later in bone development, together with high expression at sites of rapid remodelling, indicate that OPN deposited on the surface of mineralized connective tissues may provide a template for osteoclastic resorption.

Identification of the major phosphoprotein secreted by many rodent cell lines as 2AR/osteopontin: Enhanced expression in H-RAS-transformed 3T3 cells

/ar, a tumor promoter-inducible protein secreted by mouse JB6 epidermal cells, is the murine homolog of rat osteopontin, or 44 kD bone phosphoprotein. We report here that 2ar is also related to pp69, a major phosphoprotein secreted by normal rat kidney cells. Antisera raised against pp69 and against beta-galactosidase-2ar fusion proteins are able to immunoprecipitate the same major phosphoproteins, of apparent Mr 55-69 kD, secreted by several rat and mouse cell lines. The levels of secreted protein and cytoplasmic mRNA are dramatically elevated in NIH 3T3 cells transformed with the human bladder cancer T24 (H-ras) oncogene. These results and the work of Senger and colleagues (Cancer Res., 45, 5818-5823, 1985) imply that enhanced secretion of 2ar/pp69/osteopontin by transformation of a wide variety of mammalian fibroblasts and epithelial cells is often correlated with tumorigenicity.

Uptake of isolated metaphase chromosomes by mammalian cells in vitro.

Abstract In an autoradiographic investigation, it was found that isolated 3 H-thymidine-labelled metaphase chromosomes can penetrate into mammalian cells in vitro. The progressive changes in the cellular labelling patterns with time indicate that, in most cases, the ingested chromosome is degraded in the cytoplasm of the recipient cell and the chromosomal DNA is integrated into the host nuclear DNA. This integration of extracellular chromosomal DNA into the recipient cell DNA may involve DNA macromolecules rather than free nucleotides, and in most cases, the chromosomal DNA becomes randomly integrated among the chromosomes of the recipient cell. Rarely, whole extracellular chromosomes may become incorporated into a cell without being degraded, although this has not been established with certainty.

Retinoic acid induces anchorage- and density-dependent growth without restoring normal cytoskeleton, egf binding, fibronectin content and odc activity in a retrovirus- -transformed mouse cell line.

Abstract We studied the effects of retinoic acid (RA), a known tumor inhibitor, on a group of transformation-related properties expressed by a Kirsten sarcoma virus (Ki-MSV)-transformed BALB/c 3T3 (clone A31) cell line (KA31). The aim was to determine whether any one or a group of such transformation-specific changes, exhibited by this cell line, is an absolute prerequisite for its acquisition of the neoplastic state. Our study shows that RA treatment restores two important parameters of normalcy in KA31 cells: density-dependent growth inhibition, and anchorage-dependent growth. Retinoic acid also induces cell-to-substratum adhesiveness in a significant proportion of the treated KA31 cell population. However, four other markers of transformation—loss of microfilaments, lack of surface fibronectin, reduced epidermal growth factor (EGF) binding, and increased ornithine decarboxylase activity—remain unaltered by RA treatment. These transformation-specific changes, therefore, are dissociable from loss of anchorage- and density-dependent growth. It is suggested that RA may intervene in transforming protein kinase-induced cell surface changes that are involved in cellular malignancy and the loss of growth control in KA31 cells.

Effects of controlled exposure of L cells to bromodeoxyuridine (BUdR): I. Evidence for ordered gene replication during S phase☆

Abstract Fluorodeoxyuridine (FUdR)-synchronized mouse L cells were allowed to incorporate 5-bromodeoxyuridine (BUdR) at restricted intervals in the S phase and the effects of the selective incorporation of BUdR in DNA on the activities of seven randomly chosen enzymes (five dehydrogenases and two phosphatases) were analysed. Reductions to 56.9 and 83.3 % of the control levels were noted for glucose-6-phosphate dehydrogenase (G6PD) and alcohol dehydrogenase (ADH) activities respectively, when cells were exposed to BUdR during the 1st h of S. Acid phosphatase (AcP) activity was reduced to 81.9% of the control level following exposure to the analogue during the 3rd h of S. Exposure of cells to BUdR for the entire S period failed to increase the magnitude of the reductions in activity for any of these three enzymes. Alternately, when cells were allowed to synthesize DNA in the presence of thymidine for the 1st h of S and the remainder in the presence of BUdR, the activities of G6PD and ADH were comparable to those found in untreated cells. Exposure of cells to thymidine for the 3rd h of S, combined with exposure to BUdR for the preceding and subsequent hours of S, provided complete protection against the BUdR-mediated reduction in AcP activity. The activities of lactate dehydrogenase (LDH), 6-phosphogluconate dehydrogenase (6pGD), isocitrate dehydrogenase (IDH) and alkaline phosphatase (A1P) were found to be insensitive to treatment with BUdR, even when the period of analogue exposure encompassed the entire S period. Additional investigations carried out with G6PD for characterization of the nature of the BUdR effects suggest that the BUdR-mediated reductions in enzyme activities are not caused by the increased rates of degradation of the enzymes, formation of enzyme inhibitors or by the disproportionate replication of A-T base pairs during BUdR treatment. The alterations of enzyme activities appear to result from decreased rates of synthesis of enzymes in BUdR-treated cells. The results of the present study clearly suggest that pulse labelling of cells with BUdR at various intervals of the S phase may provide a useful approach for determining temporal localization of replication time of DNA segments that are critical for the synthesis or regulation of specific gene products.

Replicative differentiation of mammalian sex-chromosomes during spermatogenesis☆

Abstract In somatic cells of the male golden hamster 1 2 X and Y-chromosomes are late replicating, whereas in the male mouse late-replication can be found only in the Y-chromosome. Recent studies have demonstrated the presence of 2 discrete populations of spermatogonial cells in the Chinese and golden hamsters. One population showed sex-chromosomes replicating in exactly the same manner as in somatic cells whereas another population showed sex-chromosomes replicating synchronously with or earlier than some autosomes. Recent studies have also shown that in the golden hamster and rabbit embryos the X and Y-chromosomes replicate synchronously with autosomes before implantation and that the typical somatic patterns appear in embryos only after implantation. The present study demonstrates that unlike the somatic cells, the 1 2 X and Y-chromosome of the male golden hamster and the Y-chromosome of the mouse terminate replication simultaneously with or even earlier than some autosomes. This suggests that the change in the replication patterns of the sex-chromosomes from “late replicating” in spermatogonia to “early replicating” in spermatocytes takes place during successive spermatogonial mitoses and when 2 gametes having early replicating sex-chromosomes unite, the cells from early embryos retain the “early replicating” pattern until just before implantation when the typical somatic patterns appear again.

Cytoplasmic control of nuclear activity in preimplantation mouse embryos

Abstract Techniques have been developed for the production of heterokaryons between 2- and 4-cell embryos of mouse and adult somatic cells of various origins, using Sendai virus-mediated cell fusion. These techniques were used to study the cytoplasmic control of nuclear activity in 2- and 4-cell blastomeres. It was found that the cytoplasm of 2-cell blastomeres contained factors that inhibited RNA synthesis in previously competent mouse nuclei. These factors could also affect human nuclei and nuclei derived from malignant cells in a similar manner. Inhibitory factors were largely lost by the 4-cell stage. Although RNA synthesis by somatic nuclei in 2-cell cytoplasm was repressed, it was found that the transport of RNA from somatic nuclei was not. The cytoplasm of 2-cell blastomeres was found to contain components which activated RNA synthesis by hen erythrocyte nuclei which normally synthesize no RNA. The cytoplasm of 2-cell embryos was also found to contain factors that stimulated DNA synthesis in nuclei derived from a contact-inhibited culture which normally synthesizes no DNA.

Epidermal and transforming growth factors modulate secretion of a 69 kDa phosphoprotein in normal rat kidney fibroblasts

Our study shows that the secretion of a major non‐glycosylated, phosphoprotein of 69 kDa (pp69) is a specific marker for non‐transformed NRK‐49F cells. Treatment of NRK‐49F cells with EGF alone or with different combinations of EGF plus TGF‐β modulates the secretion of pp69, suggesting its relationship with cellular proliferation. Antibody raised against pp69 recognizes, in addition to pp69, another major phosphoprotein of 62 kDa (pp62) secreted by RR1022 and spontaneously transformed NRK‐49F cells. Immunoprecipitation of total cell lysates from both NRK‐49F and RR1022 cells with anti‐pp69 antibody detected only pp69. These observations suggest a precursor‐product relationship between pp69 secreted by non‐transformed NRK‐49F cells and pp62 secreted by transformed cells.

Lack of correlation between loss of anchorage-independent growth and levels of transformation-specific p53 protein in retinoic acid-treated F9 embryonal carcinoma cells

It has been shown that differentiated derivatives of retinoic acid (RA)-treated F9 embryonal carcinoma cells become non-malignant. In the present study it is asked whether this loss of malignancy is due to cellular differentiation. Because the ability of cells to grow in suspension correlates with in vivo tumorigenicity, we determined the time course of the loss of this property, after RA treatment, with relation to the differentiation to parietal endoderm and the acquisition of normalcy in several common transformation-specific properties of F9 cells. Our results show that pretreatment with RA for 24 h caused 80% inhibition of anchorage-independent growth in F9 cells, and this inhibition reached its highest level (98%) after pretreatment with RA for 48 h and longer. However, all other observed transformation-related properties, and the levels of plasminogen activator (marker for parietal endoderm) remained unaltered at this early post-treatment stage. These observations suggest that the loss of malignancy is a relatively early event in the biochemical pathways involved in the RA-induced differentiation of F9 cells. Furthermore, our data show that the presence of elevated levels of p53 alone may not be sufficient to maintain the anchorage-independent growth and the rapid proliferation of F9 cells.

Kirsten murine sarcoma virus-coded p21ras may act on multiple targets to effect pleiotropic changes in transformed cells.

Abstract Kirsten murine sarcoma virus (Ki-MSV) is a replication-defective recombinant retrovirus capable of transforming cells in culture. A Ki-MSV coded, 21,000-dalton protein (p21 ras ) is required for the maintenance of cellular transformation. It is unknown whether the p2l ras of Ki-MSV induces transformation by acting on multiple targets, as has been suggested for Rous sarcoma virus transformed cells, or by a single target mechanism. In order to resolve this question, we have used a normal rat kidney cell line transformed by a temperature-sensitive mutant strain of Ki-MSV ( ts KNRK), which codes for a thermolabile p21 ras to investigate the correlative aspects of the expression of several transformation-related cellular properties upon shifting from the permissive (32°) to nonpermissive (39°) temperature. Except for an altered morphology, an organized cytoskeleton, and increased adhesion to substratum, ts KNRK cells at 32° displayed similar properties to those of wild-type Ki-MSV transformants at either temperature. Upon shifting to 39°, anchorage- and density-dependent growth were restored, although the growth rate and glucose uptake were unaffected. The cells assumed a more flattened morphology, although adhesiveness did not increase significantly. Increased levels of cell surface fibronectin were observed within 48 hr post-temperature shift, although fibronectin levels comparable to that of normal rat kidney cells (NRK) were not observed until later. Epidermal growth factor (EGF) binding increased only slightly at 48 hr post-temperature shift, but did not approach EGF-binding levels of NRK cells. Cytoskeletal organization was invariant between the two temperatures. Although our results suggest a multiple target model for Ki-MSV-mediated transformation, a single target mechanism cannot totally be ruled out.