Gene therapy of rat medullary thyroid cancer by naked nitric oxide synthase II DNA injection.

BACKGROUND: Nitric oxide (NO), produced by NO synthase II (NOS II), is the main mediator of the tumoricidal action of activated macrophages. In the present study we examined the potential of the NOS II gene as a suicide gene for medullary thyroid cancer (MTC) therapy. METHODS: We orthotopically transplanted rMTC 6-23 cells into the inbred strain of Wag/Rij rats and constructed a plasmid carrying the NOS II gene under the control of the cytomegalovirus (CMV) promoter. RESULTS: Successive injections of tumor cells (Day 0) and naked DNA (Day 2) caused strong inhibition of tumor growth (50%, p < 0.05). Plasmid injection into established tumors (14-day tumors) resulted in the development of large cavities due to tumor cell destruction, with a significant reduction in tumor tissue volume (35%, p < 0.05). Adjacent quiescent tissues were unaffected. Cell death occurred by apoptosis as demonstrated by specific labeling. Macrophages and CD4+ lymphocytes were recruited in the treated tumors. However, tumor-specific T lymphocytes were undetectable in the spleen of treated rats. In control experiments using Lac Z as a reporter gene, expression of beta-galactosidase was detected in only 1% of the tumor cells. CONCLUSIONS: Despite a low gene transfer efficiency, NOS II plasmid produced a strong anti-tumor action resulting from its marked 'bystander' effect mainly due to NO production and diffusion. Therefore the NOS II gene appears to be a promising suicide gene therapy of human cancer.

Treatment of medullary thyroid carcinoma by combined expression of suicide and interleukin-2 genes.

Inherited medullary thyroid carcinomas (MTC) are aggressive and resistant to conventional chemo- and radiotherapies. We evaluated a novel strategy for treatment of MTC, combining "suicide" and interleukin-2 (IL-2) gene therapies. Tumors were produced in Wag/Rij rats by orthotopic injection of the rMTC 6-23 cell line, and/or derivatives expressing the herpes simplex virus 1 thymidine kinase (HSV1-TK) gene (rMTC-TK). Ganciclovir, a nucleoside analog selectively transformed to a toxic metabolite by HSV1-TK, totally eradicated rMTC-TK tumors in 60% of the animals. 1:1 rMTC and rMTC-TK mixed tumors were also strongly inhibited by ganciclovir (P < 0.05), indicating the occurrence of an efficient "bystander" effect in vivo. Double labelling of rMTC cell membranes and apoptotic nuclei revealed that, as with the TK+ cells, some TK- cells died by apoptosis. A 1:1 mixture of rMTC and rMTC-TK cells was administered to produce established tumors and then rMTC cells, transfected to express the IL-2 gene (rMTC-IL2), were inoculated. Combined ganciclovir and IL-2 treatment improved the inhibition of tumor growth compared to that following ganciclovir alone (86% compared to 54%, P < 0.05). This treatment also significantly enhanced macrophage activation and tumor infiltration by CD8+ and CD4+ T lymphocytes. These results open an avenue for combining suicide and immunoregulatory gene therapies for MTC management in man.

Influence of laminin substratum on cell proliferation and CALC I gene expression in medullary thyroid carcinoma C cell lines.

Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT) and CT gene-related peptide (CGRP), the two splice peptide products of the CALC I gene. Normal and hyperplastic C cells are intrafollicular, in contact with the basement membrane (BM) that is maintained around the differentiated tumors. To investigate the relationships between MTC evolution and BM constituents, we examined the modifications induced by laminin-1 and -2 (merosin), two isoforms colocalized in the follicular BM, on three MTC cell lines: murine rMTC 6-23 and CA-77 cells, and human TT cells. Laminin exerted a mitogenic activity on rMTC 6-23 and on TT cells, causing a concurrent decrease in both CT and CGRP mRNA levels and production of the peptides. Conversely, laminin reduced the proliferation rate and enhanced CGRP synthesis and secretion in CA-77 cells. This antiproliferative response, which coincides with an increase in differentiation markers, is comparable to that reported in normal cells and also in the neoplastic Caco-2 cell line. This suggests that laminin could exert opposite effects depending on the stage of tumor evolution.

Immune response against medullary thyroid carcinoma (MTC) induced by parental and/or interleukin-2-secreting MTC cells in a rat model of human familial medullary thyroid carcinoma.

The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cell engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8+ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4+ T lymphocytes were scarce. Our results suggest that the CD8+ T lymphocytes are implicated in the anti-tumour reaction elicited by the IL-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC.

Calcitonin secretion, C cell differentiation and proliferation during the spontaneous development of murine medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, synthesizes large amounts of calcitonin (CT), its biological marker. However, in some cases with a poor prognosis, MTC is associated with low basal CT levels owing to a decrease in the thyroid CT content. Using a murine model of human MTC, we studied the relationships between CT biosynthesis, C cell proliferation, and the circulating CT level during MTC progression. Cell proliferation was revealed by autoradiography of radioactive thymidine incorporation in dividing nuclei, after CT or CT mRNA detection by immunocytochemistry (ICC) or in situ hybridization (ISH). All rat thyroids showed a severe hyperplasia of C cells containing significant amounts of CT and CT mRNA, and a very low mitotic index. Tumours were found in 68% of the thyroids. In the strongly immunoreactive small nodules (ICC+), many labelled nuclei were observed. Subsequently some nodular cells, still containing detectable CT mRNA (ISH+), were not detected by immunocytochemistry (ICC-) owing to a dramatic decrease in secretory granules. Their mitotic index increased, and a rise of the basal CT plasma level was noted. These ISH+, ICC- tumour MTC cells represent a modified aggressive tumour C cell population exhibiting an increased ability to proliferate and were detected by the rise in the basal circulating CT level.

Evaluation of somatostatin biosynthesis, somatostatin receptors and tumor growth in murine medullary thyroid carcinoma.

Spontaneous medullary thyroid carcinomas (MTCs) of old rat thyroids were analyzed for the expression of somatostatin and somatostatin binding sites in tumoral C cells in relation to the stage of tumor development, the mitotic activity of tumoral tissue and calcitonin biosynthesis as a marker of C cell differentiation. High levels of both immunoreactive somatostatin and its mRNA were detected in a subpopulation of tumoral C cells, gathered in areas suggesting a clonal proliferation and located preferentially at the periphery of the tumor. These cells also displayed high levels of calcitonin and its mRNA. However, many calcitonin immunoreactive cells showed no sign of somatostatin synthesis. The proliferative activity of the somatostatin-containing areas was low and slow compared to the areas lacking somatostatin production. However, it increased during the course of tumor growth. Somatostatin binding sites, measured with in vitro receptor autoradiography using 125I-[Tyr3]-octreotide or 125I-[Leu8, dTrp22, Tyr25]SS-28, were not detected in any of the MTCs tested. In rat MTC cells, somatostatin was associated with differentiation and slow proliferation, two parameters inversely correlated with the progression of malignancy. As expected, owing to the highly regulated secretion of the differentiated endocrine cell type, its presence was correlated with low basal calcitonin levels. However, the absence of somatostatin binding sites on any type of MTC cells does not favor a direct autocrine regulation of this peptide in this murine model of human MTC.

Regulation of plasma calcium and phosphate in calcitonin-infused rats.

The effects of long-term constant infusion of moderate doses (2-32 ng/h) of salmon calcitonin (sCT) on plasma Ca (and its radionuclide 45Ca), Pi, Mg, and on endogenous rat CT (rCT) metabolism were investigated in the rat. Daily variations were included. 1) The plasma concentrations of Ca and Pi fell and that of Mg increased transiently during infusion, with the duration of responses (1-3 days) depending on the sCT dose. Rats infused with 8 ng/h sCT remained sensitive to CT after 7 and 14 days, as indicated by the effects of minipump removal and of a bolus injection of exogenous sCT on plasma mineral concentrations. 2) In contrast to control rats, the well-established daily variations in plasma Ca and Pi levels were no longer observed after 7 and 14 days of sCT infusion (8 ng/h), but normal variations persisted for plasma Mg, circulating rCT, rCT mRNA, and rCT thyroid content. 3) Statistical analysis of plasma mineral data, collected at five sequential times during days 7 and 14, showed that the means were not significantly different and that the daily variations were essentially identical on days 7 and 14 in control rats. In contrast, the variability of measurements for plasma Ca and Pi, but not for Mg, increased significantly between days 7 and 14 in infused rats, and the mean differences were significantly lower in infused rats on day 7 than in control rats. These results are consistent with a transitory loss of the daily variations for Ca and Pi (day 7) and the later (day 14) spontaneous recovery of some variations in these parameters, although the individuals remain unsynchronized.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian rhythms of calcitonin gene expression in the rat.

The daily changes in rat thyroid calcitonin and its specific mRNA concentrations, and the relationship between their dynamics and the plasma levels of calcitonin, calcium and phosphate over a 24-h period were investigated. The circulating calcitonin concentration rose during the daily dark period when plasma calcium and phosphate levels were minimal, indicating that plasma calcitonin rhythm cannot be generated directly by a linear effect of calcium on hormone secretion. Moreover, we established that the expression of the calcitonin gene also exhibited periodic dynamics observable at the pretranslational level: the gland content of hybridizable specific calcitonin RNA displayed daily rhythms; specific RNA levels peaked during the light period and were minimal during the first part of the dark period. Significant changes in thyroid calcitonin concentrations also occurred over a 24-h period. Statistical analyses which distinguished between variations over the 24-h period and residual variations were performed to test the relationships between the various parameters. The daily rhythms of hybridizable RNA, thyroid calcitonin and plasma minerals appeared to be in phase, while the plasma calcitonin concentration displayed variations out of phase with these rhythms. The implication of the correlations observed on the residual variations is discussed in comparison with the temporal relationship between the daily variations. The results fit the hypothesis that hormone production and secretion are self-oscillating processes. Plasma concentrations of calcium and phosphate might play a role in the synchronization of the calcitonin metabolism periodicity.

A physiological view of in vivo calcium dynamics: the regulation of a nonlinear self-organized system.

Our aim is neither to re-evaluate the term homeostasis, nor to summarize the conventional concepts in the field of calcium metabolism and its regulation, nor even to comment on their advantages and their limitations (excellent recent reviews have been published). This paper is rather a position article and references to the current literature will be made only if they contribute to a better understanding of our proposals; in contrast, emphasis will be placed on a literature which has, until now, remained unfamiliar to the field of calcium metabolism. The text is organized around three related features which are largely dictated by the characteristics of our recently published compartmental self-oscillatory model for rat calcium metabolism: (a) The circadian behavior associated with calcium dynamics in vivo may be viewed as a "key" temporal behavior for investigating the spatiotemporal organization of calcium metabolism in the normal rat. Within the bone, a large part of this circadian behavior should stem from the physico-chemical properties of the transformations of calcium-phosphate associations at the extracellular fluid (ECF)/mature bone interface; (b) an important part of the maintenance of a nearly constant plasma calcium concentration (homeostasis) results from interaction between nonlinear oscillators belonging to both calcium metabolism and calcium-regulating hormones. This implies that: firstly, calcium metabolism, like any biological system, is a complex dynamic system which has evolved over a long period and whose metabolic components--gut, kidney, bone--are organized as dynamic entities, adapted to periodic relationships with the external environment. The intrinsic nature of the circadian behavior of bone calcium efflux proposed here is a sufficient demonstration of this. Secondly, the existence of rhythmic variations in the main calcium regulating hormones, parathyroid hormone (PTH), calcitonin (CT) and vitamin D (VitD), are in agreement with this argument. As developed below, fascinating properties emerge from interaction between oscillators (hormones and target organs) which provide a new perspective on calcium regulation; and (c) one of the striking properties of the kind of nonlinear dynamic system required for this representation of calcium metabolism is that periodicity is only one of many temporal expressions. Thus, qualitative diversity in the temporal expression of calcium metabolism can be expected with varying experimental situations and different modes of temporal regulation of calcium metabolism might be physiologically effective, depending on the species studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Circadian variations in plasma calcium and calcitonin: effect of calcium deficiency and fasting.

Circadian fluctuations of plasma calcium and immunoassayable calcitonin levels were studied in normal and calcium-deficient 2-month-old rats. The relationship between these parameters was also studied in animals which had been fasted for short periods. The plasma calcium rhythm persisted and was even amplified in rats placed on a 4-week calcium-deficient diet. In these rats, as in normal rats, the plasma calcium concentration diminished during the dark period. Calcitonin levels increased at the onset of the feeding period in normal rats but, in calcium-deficient rats, the pattern changed completely, with a major peak at the end of the light period and remaining at a low level during the dark feeding period. This modification of calcitonin rhythmicity appeared to be dependent on the degree of calcium deficiency. Fasting had little effect on calcitonin rhythms in either normal or calcium-deficient rats. It is concluded that the calcitonin rhythm is relatively independent of feeding per se and that there appears to be no simple relationship between plasma calcium and calcitonin concentrations. It is suggested that the results may best be interpreted as reflecting the presence of rhythmic endogenous phenomena which are intrinsic to calcium metabolism and its regulation in the rat.