Transplantable Leydig Cell Tumor Research Articles

First cytogenomic characterization of murine testis tumor cell line MLTC-1

The cell line MLTC-1 was established in 1982 as a transplantable Leydig cell tumor from a C57BL/6 mouse. The cell line has already been applied in >100 studies: still, the only information about its genetic content is given in the first description: MLTC-1 initially had a polyploid karyotype. Here, a molecular karyotyping and multicolor banding-based molecular cytogenetic study was done to provide the first chromosomal/ (molecular) cytogenetic characterization of MLTC-1. A hexaploid karyotype with 72 to 105 chromosomes was hereby characterized. Besides polyploidy, unbalanced two- and three-way translocations, dicentrics and one neocentric derivative were identified. Also, no Y-chromosome could be detected in this clearly male derived cell line. Overall, a completely unbalanced genome is present in MLTC-1 with ~20 regions being subject to copy number losses or gains. After translating these imbalances into the human genome in a standardized way, a 40% match of imbalances with human Leydig cell tumors was evident; however, about the same rate of concordance was detectable for human spermatocytic seminomas and non-seminomas as well as testicular germ cell tumor. Accordingly, MLTC-1 is better suited as an advanced testicular germ cell tumor model in general, rather than specifically for human Leydig cell tumors.

Mouse Leydig tumor cells produce C-19 steroids, including testosterone

The mouse Leydig tumor cells (MLTC-1) were derived from a transplantable Leydig cell tumor carried in C57BL/6 mice. The original cell line (M5480) produced testosterone and little progesterone. However, it was later shown that there were two subtypes of the cell line, one producing mainly progesterone and termed M5480P and the other which produced androgens and termed M5480A. MLTC-1 cells are reportedly derived from the former. We studied the production of testosterone by MLTC-1 cells using a specific and sensitive testosterone RIA, tandem mass spectrometry (TMS) and examined the expression of mRNA of some key enzymes involved in steroidogenesis. Although the molar yields were 1:20:60 for testosterone, androstenedione and progesterone, respectively, in response to human chorionic gonadotropin (hCG), testosterone measured by our RIA accounted for 94% of the testosterone immunoreactivity. Both MLTC-1 and Balb/c Leydig cells expressed Steroidogenic Acute Response (StAR) protein mRNA in response to hCG. Cytochrome P450 17α-hydroxylase/17,20-lyase mRNA was expressed constitutively in MLTC-1 and Balb/c Leydig cells. Whereas the latter expressed 17β-hydroxydehydrogenase/17-ketoreductase isoform Type 3 mRNA in response to hCG, MLTC-1 cells expressed isoform Type 7 constitutively. The absence of isoform Type 3 in MLTC-1 cells thus may account for the low conversion of androstenedione to testosterone in this cell line. However, with a very specific and sensitive RIA even the low production of testosterone becomes meaningful. In conclusion MLTC-1 cells produce testosterone.

The effect of meal size on postprandial carbohydrate metabolism in normal and tumor-bearing rats.

Doubling the size of a meal causes less than a two-fold increase in the thermic effect of feeding. One possible reason for this is that larger meals may be associated with a change in the pathway of postprandial hepatic glycogen synthesis from the indirect pathway, involving gluconeogenesis, to the more energetically efficient direct pathway. We have therefore investigated the effect of meal size on the relative contributions of those two pathways both in normal rats and in tumor-bearing rats, which have previously been shown to utilize the indirect pathway to a greater extent. Rats bearing a transplantable Leydig cell tumor and freely fed controls were fasted overnight and given a test meal amounting to 12 or 24 kJ of their normal diet. They were then injected with 3H2O and 14C-glycerol and killed one hour later. The total amount of 3H incorporated into liver glycogen was not affected by meal size, although it was greater in tumor-bearing rats than controls. Analysis of the 3H labelling at different positions in the glycogen glucose residues showed that the proportion of glycogen synthesized via pyruvate, which tended to be greater in tumor-bearing rats, was significantly reduced by increasing the size of the meal. Glycogen synthesis from glycerol was not affected by either meal size or tumor growth. Increasing the size of the meal increased the rate of fatty acid synthesis in both the liver and the epididymal fat pad, but not the tumor. Thus increasing the size of the meal appeared to increase the proportion of glycogen synthesized by the direct pathway from glucose in both tumor-bearing and control animals.

Lipid metabolism in cachectic tumor-bearing rats at different stages of tumor growth.

Rates of lipogenesis and lipoprotein lipase (LPL) activity were measured in liver, adipose tissue, heart, and tumor at several stages during 10 days of palpable growth of a transplantable Leydig cell tumor in rats. This model showed the same characteristics as human cancer cachexia, including anorexia, weight loss, and muscle wasting. Comparison with pair-fed controls showed that the rate of loss of body fat was greater than could be explained by anorexia alone. The rate of lipogenesis tended to decrease during the later stages of tumor growth, particularly in the liver, where there was a statistically significant reduction on Days 5 and 10. This may be largely attributable to decreased availability of substrates caused by decreasing food intake and increasing glucose uptake by the tumor. There was a significant decrease in plasma glucose concentration by Day 10. In contrast, LPL activity in adipose tissue was depressed from the earliest stage of tumor growth, and this is likely to be a major cause of lipid depletion in cancer. There was no difference in adipose tissue LPL activity between the fed and postabsorptive states in the tumor-bearing rats, indicating that the normal response to nutrient intake was impaired. Thus, treatment of cancer cachexia should concentrate on normalizing the metabolic response to nutrient ingestion.

Hypercalcemia in association with a Leydig cell tumor in the rat: A model for tumor-induced hypercalcemia in man

The etiology of tumor-induced hypercalcemia was investigated in a transplantable Leydig cell tumor of the Fischer rat. In this model, serum calcium rose from a baseline of 10.4 ± 0.3 m mg/dl to 12.5 ± 0.4 mg/dl at day 10 and 16.4 ± 1.3 mg/dl (p<0.001) at day 13 post transplant. Urinary calcium also increased from 1.52 ± 0.17 mg/d to 3.52 ± 0.72 mg/d (Day 12, p<0.01). Serum phosphate decreased from a baseline of 7.5 ± 0.3 mg/dl to 5.5 ± 0.6 mg/dl at day 13 (p<0.05). At day 13 serum immunoreactive parathyroid hormone levels fell 76% from baseline (p<0.01). Calcitonin increased from 59 ± 2 pg/ml to 88 ± 9 pg/ml (p<0.01). The plasma prostaglandin E metabolite, 13, 14-dihydro-15-keto-PGE 2 increased from 407 ± 103 pg/dl to 647 ± 62 pg/ml (p<0.05) and the active Vit D compound 1, 25(OH) 2D increased from 94.8 ± 5.2 pg/ml to 162.3 ± 11.8 pg/ml (p<0.01). Urinary cyclic AMP did not decrease in parallel with the parathyroid hormone level and, in fact, increased from 146 ± 3 nmol/d to 172 ± 27 nmol/d (NS). Administration of the cyclooxygenase inhibitor indomethacin (20 mg/Kg/d) or hydrocortisone (50 mg/Kg/d) did not prevent the development of hypercalcemia. This model is similar to many patients with humoral hypercalcemia of malignancy who demonstrate suppression of parathyroid hormone with elevated urinary cyclic AMP excretion and may prove useful in the understanding of the responsible mechanisms.

Degenerative changes in the digestive glands of rats bearing Leydig cell tumors.

We undertook this study to determine the effects of a transplantable Leydig cell tumor on the major digestive glands of the host rats. It has been known that after bearing this tumor for only two weeks, the rats become anorexic and cathectic, develop hypercalcemia and osteolysis, and their peripheral bone marrow becomes hyperplastic. We now demonstrate that the parotid glands undergo marked degeneration including depletion of secretory product, apparent loss of acinar organization and the appearance of conjoined nuclei. The submandibular and sublingual glands and the pancreas are virtually unaffected. The liver undergoes fatty degeneration, the Kupffer cells become more prominent and more numerous, and the sinusoids sometimes contain small islets of hemopoietic tissue.

Tumor-induced anorexia in the Wistar rat.

The transplantable Leydig cell tumor of Wistar rats, LTW(m), caused decreased food consumption and weight loss in the host within 2 weeks of implantation. The tumor was small, did not metastasize, and did not affect several parameters of biochemical function. When the tumors were removed, increases in food intake and body weight occurred within 72 hours and were sustained. Reimplantation of tumors caused anorexia to recur. Parabiotic pairs of rats with tumor in one partner also experienced weight loss. Those rats in parabiosis with tumor-bearing rats gained less weight than those in parabiosis with control rats. These observations suggest that the LTW(m) tumor causes anorexia and that this anorexia is mediated by a circulating substance.

Inhibition of anterior pituitary hormone release by infusion of calcium chloride in the rat.

We have previously demonstrated that hypercalcemia in association with a transplantable Leydig cell tumor caused marked alterations in the hypothalamic-pituitary axis of the rat. To determine if the alterations were the result of hypercalcemia rather than other tumor-related factors, we studied the effects of hypercalcemia on the neuroendocrine axis by infusing varying concentrations of CaCl2 into the femoral vein of rats. Three groups of 40 mature male rats each received TRH (10 μg/kg), haloperidol (H; 0.5 mg/kg), or LHRH (10 μg/kg) through an indwelling cannula in the right common carotid artery 30 min after calcium infusion was initiated. Blood samples (100 μl) were drawn through the cannula 30 min before, during and 15, 30, and 45 min after TRH, LHRH, and H. Basal TSH, PRL, and LH levels were suppressed after calcium infusion, and hormonal responses to each of the releasing agents were blunted proportionally to the degree of hypercalcemia achieved. There were significant negative correlations (r = −0.72 to r = −0.83; P < 0.01) between serum calcium levels and TSH responses to TRH, PRL responses to TRH and H, and LH responses to LHRH at the time of administration of the releasing agents. Anterior pituitary hormone secretion of TSH, PRL, and LH was suppressed proportionally to the magnitude of hypercalcemia achieved in the rat. Thus, it appears that serum calcium levels should be taken into account when evaluating the hypothalamicpituitary axis. (Endocrinology106: 1809, 1980)

Growth and composition of a transplantable murine Leydig cell tumor.

C57BL/6J mice inoculated sc with 50 mg of a transplantable Leydig cell tumor (M5480) demonstrated a reproducible pattern of slow tumor growth up to day 10 followed by rapid growth at the rate of approximately 0.5 g per day through day 27. The mean survival time of tumor-bearing mice was 33 days, when tumor weight accounted for more than 25% of total body weight. The rapid increase in weight occurring around day 10 resulted largely from a 20-fold increase in the quantity of extravasated blood inside the tumor, which in turn promoted a 50% reduction in host hematocrit. Sustained enlargement of M5480 during the third and fourth weeks of growth was supported by proliferation of tumor cells. Apart from blood-filled cavities, over 90% of the tumor consisted of neoplastic Leydig cells exhibiting generalized cytoplasmic features usually associated with mitotic activity. An activated macrophage was the next most abundant cell type, accounting for 2-3% of the nucleated cell mass. The remaining 3% was occupied by vascular elements, leukocytes, and giant cells. Depending on the age of the tumor, varying proportions of the cell population showed signs of anoxic degeneration. Degenerate cells were minimal at day 14, accounting for less than 4% of the total population, and maximal beyond day 21, when they occupied more than 50% of the cell mass.

Hypercalcemia and neoplasia: a model system.

A transplantable Ley dig cell tumor of the Fischer rat produces hypercalcemia and hypercalciuria. The behavior of this tumor has been studied in individual rats and groups of rats. Continued hypercalcemia leads to hypophosphatemia and azotemia and an eventual decrease in serum calcium and calcium excretion. A 23 factorial design elucidates the effects of tumor, castration, thyroparathyroidectomy and the interactions of these procedures on the level of serum calcium and excretion of calcium in the urine. The greatest changes in serum calcium and calcium excretion in the urine are seen in castrated and/or thyroparathyroidectomized groups of rats. These observations suggest a role for gonadotropins and thyrocalcitonin in the achievement of hypercalcemia. The effects of this tumor appear to make it an ideal model system for studying some forms of hypercalcemia associated with localized neoplasia. (Endocrinology 88: 1210, 1971)

Biological features of a transplantable Leydig cell tumor in rat.

Biological features of a transplantable Leydig cell tumor in rat.