Effects of hormones on growth and alpha-lactalbumin activity in the transplantable rat mammary tumor MCCLX.

We have examined the growth and differentiation properties of the transplantable mammary tumor line of the A X C rat, MCCLX. The MCCLX tumor grows rapidly and exhibits high activity of the milk protein alpha-lactalbumin (alpha LA) (40% of the level in midlactating mammary gland) during serial passage in estrogen-supplemented (E+) intact males. Hypophysectomy of hosts bearing established tumors results in rapid tumor regression and rapid loss of alpha LA protein activity. Treatment with ovine prolactin three times daily (total of 2 mg/day) completely restores alpha LA activity and results in a partial restoration of tumor growth. In females, MCCLX grows only in E+ virgins or in pregnant hosts. During lactation, tumor growth ceases. However, alpha LA activity is equally high in E+, pregnant, and lactating females. Behavior of MCCLX apparently depends on circulating lactogenic hormones. When hormone levels are very high, as in E+ intact males or pregnant females, both growth and alpha LA activity are stimulated. In lactating hosts, having lower serum lactogen, alpha LA activity could be maintained in the absence of growth. In hypophysectomized hosts, even with continuous estrogen supplementation, alpha LA activity disappears and growth ceases, although ovine prolactin can replace the pituitary in restoring these traits. MCCLX contains prolactin-binding capacity; thus, direct action of the lactogenic hormones on the tumor is possible. Our results indicate that MCCLX is a stably hormone-responsive tumor in terms of growth and differentiation.

Effects of phosphatidylethanolamine and phosphatidylcholine in membrane phospholipid on binding of phorbol ester in rat mammary carcinoma cells.

Mammalian cells in culture can be classified as either ethanolamine (Etn)-responsive or Etn-nonresponsive with regard to their growth. Epithelial cells and some of their transformed derivatives are the Etn-responsive type. When these cells are grown without Etn, the content of membrane phospholipid becomes significantly altered. Namely, the content of phosphatidylethanolamine is reduced and that of phosphatidylcholine is increased. In addition, the growth rate of these cells is reduced. Therefore, it is likely that the phosphatidylethanolamine deficiency or phosphatidylcholine excess is unsuitable for some membrane-associated functions resulting in the cessation of growth. In order to test the above hypothesis, we examined the binding of a tumor-promoting phorbol ester, [3H]phorbol 12,13-dibutyrate (PDB), to an Etn-responsive rat mammary carcinoma cell line 64-24 grown with (Etn-plus) or without Etn (Etn-minus). The time course of binding was very similar between Etn-plus and -minus cells, except that the level of saturation was higher in Etn-plus cells, whereas the time course of chase of the bound PDB was significantly different between the two types of cells. Both types of cells have one class of binding sites for PDB. The dissociation constant (Kd) for [3H]PDB in Etn-plus cells was 34.0 nM and the number of binding sites at saturation was 2.7 x 10(12)/mg protein or 3.6 x 10(5)/cell. The corresponding values in Etn-minus cells were 61.4 nM and 3.2 x 10(12)/mg protein or 5.4 x 10(5)/cell, respectively. Although the difference in Kd values of the two types of cells was only 2-fold, this difference was statistically significant. On the other hand, the number of binding sites/mg protein in these cells was very similar. Since the amount of protein/cell was 1.4-fold higher in Etn-minus cells as compared to that of Etn-plus cells, the number of binding sites/cell was larger in Etn-minus cells. PDB affected the rate of proliferation of 64-24 cells differently, depending on whether they were grown in the presence or absence of Etn. These results suggest that the phosphatidylethanolamine and/or phosphatidylcholine content of the membrane phospholipid affects cellular functions mediated by phorbol esters.

Receptor characteristics of the rat mammary carcinoma cell line 64-24.

Saturable binding of androgens, glucocorticoids, and triiodothyronine was found in the 64-24 hormone-responsive rat mammary carcinoma cell line. Androgen receptors had a dissociation content (Kd) for methyltrienolone of 3.4 X 10(-10) M and a binding capacity of approximately 10,000 sites/cell in whole cells. 5 alpha-[3H]dihydrotestosterone (DHT) was specifically taken up into approximately 2,150 nuclear sites with an affinity of 8.3 X 10(-10) M when nuclei were isolated from whole cells incubated with [3H]DHT. Sucrose gradient centrifugation of cytosol prepared from these cells revealed a displaceable [3H]DHT-binding component which migrated at 8S. Sedimentation analysis with high salt gradients of nuclear extracts from cells incubated with [3H]DHT revealed a peak of radioactivity in the 4S region which was abolished by coincubation of the cells with excess nonradioactive methyltrienolone. Receptors for [3H]dexamethasone were more abundant (approximately 50,000 sites/cell) in whole cells and had a Kd of 7.5 X 10(-9) M, but the number of nuclear binding sites was similar to that for androgens. Specificity studies using unlabeled steroids showed that each of the two classes of steroid receptors had greater affinities for their appropriate hormones. High affinity receptors for estrogens and progestins were not detectable in these cells. Triiodothyronine receptors were demonstrable but at a very low binding capacity (1,100 sites/cell). The Kd of these receptors was 0.6 X 10(-10) M. Cytogenetic studies revealed 44 chromosomes/mitosis with several unique markers. These receptor and karyotypic features suggest that the 64-24 cells may be useful in studying androgen action on breast cancer independently of estrogen or progestin influence, as well as the effects of thyroid hormone and glucocorticoids on breast cancer cells.

Intra-strand biases in bacteriophage T4 genome.

In bacteriophage T4, a major portion of DNA replication is initiated at random along the map, although several proven and putative origins have been described for early replication. In order to analyze the contribution of transcription and translation as well as DNA replication to intra-strand bias from A = T and G = C, we examined the pattern of the intra-strand biases in the first, second, and third codon positions of the coding regions as well as the intergenic regions of the T4 genome. We found, along the map, characteristic biases both from A = T and G = C for each codon position and the intergenic regions. The bias patterns were closely associated with the location of the sense and anti-sense segments in the genome. The results suggest that: (1) transcription-associated mutation is likely a significant cause of the bias, which is suggested by the pattern of the AT bias (bias from A = T) in the third codon position; (2) DNA replication coupled bias may also exist, which is suggested by the pattern of the GC bias (bias from G = C) in the third codon position and the intergenic regions; and (3) the bias patterns of the first and second codon positions of the sense segments are consistent with universal properties of the coding sequence that G is in excess and T is deficient in the first codon position, and G is deficient in the second codon position.

Growth responsiveness to prolactin and its loss in normal rat mammary cells in culture.

Growth response of mammary epithelial cells to hormones, particularly to prolactin, was studied by using a primary culture of rat mammary gland organoids. After allowing the cells to attach and spread on the surface of plastic culture dishes, the effect of hormones was tested by means of [3H]-thymidine incorporation and autoradiography in a medium containing 1% fetal calf serum. In this system, prolactin showed a modest but significant growth-stimulatory activity (50% over control), and addition of insulin or hydrocortisone or both enhanced the growth stimulation of prolactin to a large extent. Growth stimulation caused by these hormones without prolactin was always significantly lower than that caused with prolactin. A dose-response study indicated that prolactin can stimulate growth at physiological concentrations. The maximum stimulation was observed at 1-5 micrograms/ml. The growth-stimulatory effect of prolactin was decreased as the culture period was prolonged, and by the 4th day in culture the effect was no longer observed. In contrast, the stimulatory effect of insulin was constant over the 5-day culture period. Phosphoethanolamine, which has been shown to be a growth factor for some rat and human mammary carcinoma cells, showed 2-fold growth stimulation when added with prolactin, insulin or hydrocortisone. The stimulatory effect was again not observed in older cultures, as in the case of prolactin.

Phosphatidylethanolamine deficiency in membrane lipids inhibits keratinocyte intercellular networks formation.

Ethanolamine (Etn) is required for the growth of epithelial cells in culture. Without Etn, the amount of phosphatidylethanolamine (PE) in membrane lipids is reduced, and cell proliferation stops. When the membrane lipids are deficient of PE, some extracellular signaling processes become impaired. In this study, we examined the effect of Etn deprivation on the formation of intercellular networks in immortalized human oral keratinocytes. Keratinocytes proliferate with undifferentiated morphologies in a low-calcium medium, whereas they undergo differentiation to form intercellular networks in a high-calcium medium. The cells were first cultured with or without Etn supplement in a low-calcium (0.07 mM) medium, and then the calcium concentration was raised to 1.8 mM. The localization and organization of the following proteins were examined: (1) desmogleins and plakoglobin in desmosomes, (2) E-cadherin and beta-catenin in adherens junctions and (3) actin and keratin filaments in cytoskeletons. As expected, in the Etn-supplemented cells, the elevated level of calcium induced the junctional localization of the proteins associated with desmosomes and adherens junctions and also induced the formation of keratin and actin networks. On the contrary, in the Etn-deprived cells, the elevated level of calcium induced none of the above processes. The results suggest that having a sufficient amount of PE or proper phospholipid composition in the membranes is crucial for differentiation in epithelial cells.

Insufficiency of transformation by simian virus 40, polyomavirus, EJ-ras, or v-myc oncogenes for conversion of ethanolamine-responsive mammary cells to ethanolamine-nonresponsive cells.

Normal mammary epithelial cells (ethanolamine responsive) require ethanolamine to enable them to grow in defined culture medium because they cannot synthesize de novo a sufficient amount of phosphatidylethanolamine. Mammary tumor cells which retain properties of the normal tissue are also likely to be ethanolamine responsive, whereas dedifferentiated, highly tumorigenic mammary tumor cells are ethanolamine nonresponsive. The nonresponsive tumor cells are able to synthesize the necessary amount of phosphatidylethanolamine to sustain growth. Therefore, the progression of malignancy seems to convert ethanolamine-responsive mammary cells to ethanolamine-nonresponsive ones. In an attempt to prove the above assumption and to understand the mechanism responsible for the conversion during the progression of malignant transformation, mammary tumor cell line 64-24, which is typically ethanolamine responsive, was transfected with simian virus 40, polyomavirus, EJ-ras, or v-myc oncogenes, and the resulting transfectants were examined for their growth response to ethanolamine. Many of the transfectants exhibited typical transformed phenotypes; however, none of the transfectants converted to ethanolamine-nonresponsive cells. Some of the SV40 and polyomavirus transformants were able to grow in the absence of ethanolamine, although they grew better in the presence of ethanolamine, unlike typical ethanolamine-nonresponsive cells. These cells could grow in the absence of ethanolamine, even though their membrane phospholipid was phosphatidylethanolamine deficient. The present study indicates that the expression of any one of the four oncogenes tested, which allows the cells to exhibit transformed phenotypes in 64-24 cells, is not sufficient for the conversion of ethanolamine-responsive cells to -nonresponsive cells.

Abnormal function of protein kinase C in cells having phosphatidylethanolamine-deficient and phosphatidylcholine-excess membranes.

When rat mammary carcinoma 64-24 cells are grown in the absence of ethanolamine, their membrane phospholipid composition changes significantly, becoming phosphatidylethanolamine-deficient and phosphatidylcholine-excess due to a reduced de novo rate of phosphatidylethanolamine synthesis, and growth stops. We have assumed that this membrane phospholipid environment is not suitable for membrane-associated functions. We have previously demonstrated that functions normally stimulated by tumor-promoting phorbol ester, phorbol 12,13-dibutyrate, are not stimulated in ethanolamine-deprived cells, suggesting that function of protein kinase C may be abnormal under the altered membrane environment. In the present study, the behavior of protein kinase C in 64-24 cells grown in the presence and absence of ethanolamine (having normal and phosphatidylethanolamine-deficient/phosphatidylcholine-excess phospholipid) was compared by enzyme assay as well as Western blotting. The results show that the nature of association of protein kinase C to the membrane, which is induced by phorbol ester, is abnormal when cells have the altered membrane phospholipid, and thus argue that membrane phospholipid environment is important in the function of protein kinase C.

A rat mammary carcinoma in vivo and in vitro: establishment of clonal lines of the tumor.

Clonal cell lines from a pituitary hormone-dependent rat mammary carcinoma were established. The growth of these cell lines in vitro is markedly stimulated in the presence of prolactin. The cells grow well in a medium supplemented with fetal-calf serum (generation time: 9-10 hr), but do not grow with calf serum. Fetal-calf serum supports little growth when the serum is subjected to anti-prolactin-affinity chromatography. Gel filtration of fetal-calf serum indicates that the growth stimulatory activity is in a large molecular weight material (close to 100,000), suggesting that prolactin is associated with another serum component. A variant of the original tumor, which does not require hormone for growth, was obtained, and clonal cell lines of this tumor were also established. These cells, in contrast to the hormone-dependent ones, grow well with either fetal-calf serum (with or without anti-prolactin treatment) or calf serum.

Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.

Phosphatidylethanolamine biosynthesis in rat mammary carcinoma cells that require and do not require ethanolamine for proliferation.

Epithelial cells and some of their transformed derivatives require ethanolamine to grow normally in defined culture medium. When these cells are cultured without ethanolamine, the amount of cellular phosphatidylethanolamine is considerably reduced. Using a set of rat mammary carcinoma cell lines whose growth is responsive (64-24 cells) and not responsive (22-1 cells) to ethanolamine, the biochemical mechanism of ethanolamine responsiveness was investigated. The biosynthesis and metabolism of phospholipid, particularly of those involving phosphatidylethanolamine, were thus compared between the two types of cells. The incorporation of [3H]serine into phosphatidylserine and phosphatidylethanolamine in 64-24 cells was 60 and 37%, respectively, of those in 22-1 cells. However, the activity of phosphatidylserine decarboxylase was virtually the same in these cell lines. When these cells were cultured in the presence of [32P]phosphatidylcholine and [32P]phosphatidylethanolamine, the rate of accumulation of 32P-labeled phosphatidylserine from the radioactive phosphatidylethanolamine was considerably reduced in 64-24 cells compared to that in 22-1 cells, although the rate of synthesis of phosphatidylserine and phosphatidylethanolamine from the radioactive phosphatidylcholine was similar between the two cell lines. The rate of labeling phosphatidylcholine from the radioactive phosphatidylethanolamine was also reduced in 64-24 cells, although the difference was not as great as that of phosphatidylserine. Incorporation of 32P into phosphatidylethanolamine was correlated with the concentration of ethanolamine in the culture medium in 64-24 cells, whereas in 22-1 cells the incorporation was not influenced by ethanolamine. Enzyme activities of the CDP-ethanolamine pathway were not significantly different between the two cell lines. The rate of degradation of phosphatidylethanolamine was also similar in these cell lines. These results show that ethanolamine responsiveness of 64-24 cells, and probably other epithelial cells, is due to a limited ability to synthesize phosphatidylserine resulting from a limited base-exchange activity utilizing phosphatidylethanolamine.

Leucine tRNA and cessation of Escherichia coli protein synthesis upon phage T2 infection.

The involvement of tRNA in cellular differentiation has been tested by analyzing aminoacyl-tRNA of Escherichia coli after phage T2 infection. One or two minutes after infection, half of one of the five leucine tRNA components (Leu-tRNA(1), CUG responding) undergoes a drastic structural change which leads to inactivity of both leucine acceptor activity and codon response. Whether or not the modification causes cessation of host protein synthesis without inhibiting phage-specific protein synthesis has been examined by analyzing polysome-bound leucine tRNA of E. coli before and after the phage infection. The results presented in this paper indicate that the amount of Leu-tRNA(1) used after infection was greatly reduced as compared to that used in noninfected cells. Studies of the in vitro protein-synthesizing system show that T2 mRNA rarely contains the CUG codon. A mechanism by which host mRNA translation is inhibited by the phage infection is proposed from this available information.

Steroid hormone receptors in MCCLX, a transplantable lactogen-dependent mammary tumor of the rat.

MCCLX is a transplantable rat mammary tumor which, for sustained growth, requires the elevated levels of circulating lactogen provided by pregnancy or the implantation of an estrogen pellet. High affinity receptors for estradiol, as well as for the glucocorticoids, dexamethasone and triamcinolone acetonide and the progestin R5020 were measured in the cytosols of these tumors. Estrogen binding capacities were significantly lower in the cytosols of tumors from estrogen pellet treated animals compared with tumors from pregnant animals. Ligand exchange assays demonstrated that nuclei of tumors from estrogen-treated rats contained 3-4 times the estrogen receptors but that there was a definite decrease in total estrogen binding capacity compared with tumors from pregnant rats. It was concluded that this lactogen-dependent tumor contains steroid receptors with molecular properties similar to those of normal target tissues, including estrogen receptors capable of nuclear translocation, the levels of which are modulated by the specific growth conditions.

Analysis of cytosolic phosphoethanolamine and ethanolamine and their correlation with prognostic factors in breast cancer.

Availability of accurate prognostic factors is vital in making decisions on cancer therapy. We have measured the cytosolic contents of phosphoethanolamine and ethanolamine in tumor tissues of 53 breast cancer patients in an attempt to explore the possibility that these amines could be used as prognostic indicators. The levels of phosphoethanolamine and ethanolamine were determined by high-performance liquid chromatography. The ratios of the molar quantity of these amines or amino acids to that of alanine plus tyrosine, which eluted as a single peak, were used to analyze and compare the results among different tumor samples. The results indicated that the values for phosphoethanolamine or ethanolamine varied significantly more than the values for amino acids, such as glycine plus threonine or glutamine plus serine (these amino acids were eluted as single peaks, respectively). The values for phosphoethanolamine, ethanolamine, and phosphoethanolamine plus ethanolamine were analyzed in relation to several commonly used prognostic factors of breast disease. The results indicated that groups having higher mitotic indices had significantly higher values for phosphoethanolamine or phosphoethanolamine plus ethanolamine than the group having lower mitotic indices. As the stage of the disease increased, the values for phosphoethanolamine plus ethanolamine also seemed to become higher. No correlation, however, was observed between steroid hormone receptor positive and negative groups or between positive and negative groups with regard to involved axillary lymph nodes. The content of phosphoethanolamine or phosphoethanolamine plus ethanolamine in cytosol therefore seems to be correlated with some prognostic indicators.

Growth stimulating activity in bovine pituitary extract specific for a rat mammary carcinoma cell line.

We report the isolation of a bovine pituitary growth factor (MGF) for a rat mammary carcinoma cell line, 64-24, which was isolated from a highly hormone-dependent mammary tumor. The MGF has been partially purified by a series of Diaflo ultrafiltration membrane sievings, isoelectric focusing and Sephadex columns. The MGF has a molecular weight of approximately 1,000 to 2,000 daltons and has a U.V. absorption spectrum typical for a polypeptide. Its isoelectric point is approximately pH 3.8-4.0. The factor is heat stable. The growth stimulating activity of the MGF does not stimulate other rat cell lines (22-1, RMG or HTC lines) but is specific for the 64-24 mammary tumor cell line. The MGF is not among previously reported pituitary hormones or growth factors.