Lidia Bardella

Multiple mechanisms of iron-induced ferritin synthesis in HeLa cells

Iron administration to HeLa cells stimulates the accumulation of H-subunit and L-subunit rich isoferritins at similar extent. The increase in both types of isoferritins is accompanied by an increase in the amount of messenger RNAs specific for H and L subunits. The increase in the amount of these messenger RNAs, which occurs in the nucleus as well as in the cytoplasm, is proportionately lower than the increase in the protein. These results, together with analysis of transcription in isolated nuclei indicate the existence of a mechanism of transcriptional control of ferritin synthesis, associated to the translational control described so far.

Mechanisms of regulation of ferritin synthesis in rat liver during experimental inflammation

Liver slices of turpentine-treated rats were incubated in vitro and used as a model to study synthesis and secretion of proteins during the acute-phase response. The synthesis and secretion of typical acute-phase proteins increased after treatment. Similarly, ferritin increased at 24-48 hr after treatment. Serum ferritin showed a slight and transient increase at 6 hr; however, no ferritin was detectable in liver slices medium, indicating no or negligible secretion by this tissue. Northern blot analysis of RNA extracted from total liver homogenate and from free and membrane-bound polyribosomes revealed that turpentine treatment stimulates ferritin synthesis at the translational level, possibly increasing the amount of ferritin mRNA on membrane-bound polysomes.

Regulation of ferritin synthesis in malignant and non-malignant lymphoid cells

The different amounts of H-rich and L-rich isoferritins found in malignant and non malignant lymphoid cells are accompanied by proportional variations in the relative quantity of messenger RNAs for the H and L subunits of ferritin. The correlation between levels of messenger RNAs and proteins suggests that the amount of messenger RNA plays an important role in ferritin biosynthesis in these cells. The enhanced expression of ferritin messenger RNAs in some neoplastic cells is not caused by gross alterations in the structure of ferritin genes.

FUNCTIONAL CHARACTERIZATION OF A HUMAN DNASE-LIKE PROTEIN ENCODED BY A GENE POSITIONED IN XQ28

Xib, a gene recently reported to reside on the q28 region of the human X chromosome [Pergolizzi et al. (1996) Gene 168, 267-270], contains an open reading frame homologous to those of the DNase I family enzymes. The full open reading frame of this gene has been fused to the E. coli gene of the maltose binding protein and expressed in bacteria as a chimeric protein. The partially purified chimeric protein is enzymatically active. It introduces single and double stranded breaks into supercoiled DNA, at 30 degrees C in the absence of divalent cations and at a pH optimum of 5.2. To our knowledge this enzyme represents the first cloned human endonuclease with characteristics similar to those of acidic DNase II.

Interaction of heat with chemotherapy in vitro: effect on cell viability and protein synthesis in human and murine cell lines.

Cell survival in response to doxorubicin (Dx) and cis-diammine-dichloroplatinum (cis-Pt) administration, either alone or combined with hyperthermic treatment, was analyzed in human osteosarcoma (U-2-OS), murine melanoma (B16V) and murine leukemia (P388) cell lines and in Dx-resistant sublines derived from B16V and P388. In all cell lines tested there was an enhancement of drug toxicity by hyperthermia. In U-2-OS, the increase was more pronounced for cis-Pt than for Dx. In B16V and in P388, the increase in Dx toxicity was of the same degree in Dx-senstitive and Dx-resistant sublines, whereas heat-induced sensitization to cis-Pt was higher in Dx-resistant sublines than in their Dx-sensitive counterpart. Analysis of the protein pattern in the various cell lines showed that the synthesis of heat-shock proteins induced by heat was not influenced by the combined use of drugs and heat. Moreover, in spite of some differences in the overall protein pattern, no significant differences in the basal levels of heat-shock protein synthesis or in the extent of its induction after heat shock were observed between murine cell lines relatively sensitive to Dx and their corresponding selected resistant cells.

Lack of TdT and immunoglobulin and T-cell receptor gene rearrangements in Hodgkin's disease.

To study the pathogenesis of Hodgkins disease (HD), which today remains obscure, we have undertaken a combined experimental approach: determination of TdT and molecular analysis of rearrangements of immunoglobulin heavy chain (IgH), T-cell receptor (TCR) beta chain and the T-cell rearranging gamma (TRG) genes. TdT determination indicate would the presence of immature cells that are not detected in the normal lymphnode; molecular analysis of the rearrangements of these genes would reveal the presence of even a small monoclonal population of both T and B lineages in the lymphnodes. We believe that the combination of these two types of analysis can indicate whether an expanding lymphoid clone is responsible for this disease. TdT determination was negative in all 41 cases tested. Gene rearrangements were studied in 10 cases for IgH and TCR beta genes and in 5 cases for the TRG gene. No abnormal band beside the germ-line ones was detected in any of our cases, ruling out the presence of a minor neoplastic population. We can explain these results in at least three ways: first, the neoplastic population could represent less than 1% of the total, thus escaping detection by current techniques; second, the neoplastic population is not lymphoid in nature or is composed of mature cells that do not rearrange Ig and TCR genes and therefore belongs to a true non-B, non-T lineage; third, the pathogenesis of HD is completely different from that of non-Hodgkins lymphomas (NHL) and does not involve the clonal expansion of a cell frozen at a particular maturative stage as is thought to happen in most NHL.

Constitutive and induced synthesis of heat shock proteins in transplantable hepatomas.

The synthesis of heat shock proteins (HSP) was studied in rat liver and in a series of transplantable Morris hepatomas with different growth rates, subjected to heat shock in vivo and in vitro. Different from the liver, hepatomas synthesized HSP constitutively, i.e., also before exposure to heat. This constitutive synthesis was low and limited to one HSP in the slowest-growing tumor, more marked and involving other HSP in the intermediate- and fast-growing hepatomas. In tumor that synthesized HSP constitutively, the induction of HSP in response to heat was proportionately reduced. These patterns of reaction were essentially similar in vivo ad in vitro. The amount of HSP 68 was well correlated to the levels of its mRNA in liver and in all hepatomas, whereas the increase in HSP 89 was accompanied by a corresponding increase in the related mRNA in liver and in slow-growing hepatoma, not in the other tumors, thus suggesting a different mechanism of control of HSP 89 synthesis in the more malignant hepatomas.

Synthesis of heat-shock proteins and tumor growth.

Exposure to hyperthermia induces the preferential synthesis of a set of proteins, known as heat-shock proteins. The synthesis of heat-shock proteins has been studied in rat liver cells, and human lymphocytes, and in their neoplastic counterparts. Tumor cells synthesize heat-shock proteins essentially as their normal controls, but the response of ascites hepatoma cells depends on the presence of glucose in the medium. Solid hepatoma slices seem to synthesize some heat-shock proteins constitutively, i.e., before exposure to high temperature. Any possible interpretation of this fact must take into account the growth of tumor cells.

Constitutive and Induced Expression of Heat Shock Proteins During Liver Carcinogenesis and in Hepatomas

Exposure of cells to heat or other stresses1 induces in the chromosomes the appearance of a new set of puffs2 and causes changes in gene structure and regulation that finally lead to the activation of synthesis of a distinct group of proteins. These proteins, known historically as heat-shock proteins (hsps), are now often referred to as stress proteins3–5. The best evolutionarily conserved heat-shock protein, hsp 70, is coded by a multigene family whose members respond in different ways to temperature and are subject to different regulatory mechanisms; the heat-inducible and constitutive proteins have sometimes been confused4 At least one of the members of the hsp 70 group is also growth-regulated6–7 and the activity of the corresponding gene is induced by viral and cellular oncogenes8,10. In previous studies we have examined the synthesis of hsp in injured liver cells11 and in some hepatomas of different growth rates12, and have shown that in these tumours hsp 89 and hsp 70 are expressed constitutively, while induction by heat, at least in the case of hsp 70, is progressively reduced from the slow to the fast-growing hepatomas13. Later on we became aware of the fact that hsc 73, the major heat-shock related protein, rather than hsp 70 itself, is the main constitutively synthesized protein in unstressed cells; moreover recent data showed that hsc 73 is expressed at higher level in transformed cells than in their non-transformed counterparts14. In the present paper we report the results of an investigation with a probe for hsc 73, which became recently available to us, on the constitutive expression of hsc 73 gene in liver cells submitted to a multistep carcinogenic treatment and in transplantable hepatomas, both solid and in ascitic form, of various growth rates. The main purpose of the investigation was to see if the constitutive expression of hsc 73 is an event occurring during the carcinogenic treatment, presumably associated with initiation and/or promotion, or a relatively late outcome during the growth of the established tumour, presumably associated with tumor progression.