Tumor Promoter Tetradecanoyl Phorbol Acetate Research Articles

Tumor promoter 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) can reduce the Ca‐transporting ability of Ca‐lonophores in T lymphocytes: The involvement of intracellular heavy metal lons

12-0-tetradecanoyl-phorbol-13-acetate (TPA) can significantly reduce the Ca-ionophore-induced rise in the intracellular calcium concentration (Cai) of T lymphocytes measured by quin2 or fura-2 fluorescence. This counteraction of TPA is maximal at a preincubation of 90 min at TPA concentrations higher than 20 nM. 45Ca uptake and efflux measurements directly indicate that TPA does not activate the calcium extrusion systems in thymocytes but impairs the Ca-transporting ability of Ca-ionophores. TPA causes no immobilization of the Ca-ionophores as it is demonstrated by the lack of significant changes in fluorescence and fluorescence polarisation of A23187 during TPA incubation. Similarly the energy transfer between the Tyr, Try groups of membrane proteins and A23187 shows no significant difference in control and TPA treated thymocytes. This indicates that A23187 is not in a membrane protein-bound form after TPA preincubation. The intracellular heavy metal chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) restores the ionophoretic ability of Ca-ionophores in TPA pretreated cells to the control level. Diacyl-glycerols also impair the Ca-transporting ability of Ca-ionophores. TPEN prevents this effect as well. These findings suggest that TPA and diacyl-glycerols may cause an increase in the availability of intracellular heavy metal ions. Our results may reflect a new, physiologically important mechanism of the action of diacyl-glycerols and phorbol esters.

Cloning and sequence of a secretory protein induced by growth factors in mouse fibroblasts

From a collection of 80 cDNAs representing genes that are induced by serum growth factors in quiescent mouse fibroblasts we have characterized a clone, N51, and demonstrated that it codes for a precursor of a secretory protein. N51 mRNA is rapidly induced by serum, including in the presence of cycloheximide, demonstrating that its induction does not require de novo protein synthesis. The mRNA has a short half-life of approximately 10–15 min and is dramatically prolonged in the presence of protein synthesis inhibitors. Purified growth factors such as platelet-derived growth factor and bombesin are strong inducers of N51 expression. However, epidermal growth factor, fibroblast growth factor, and the tumor promoter tetradecanoyl phorbol acetate have a negligible effect. Transcriptional analysis demonstrates that the increase in N51 mRNA levels after serum stimulation is in part due to the activation of gene transcription.

Reactive oxygen in the tumor promotion stage of skin carcinogenesis

Exposure of isolated SENCAR mouse epidermal cells to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) in vitro resulted in the production of oxidant species detected as chemiluminescence. This oxidant response can be inhibited by superoxide dismutase and copper complexes but not catalase or scavengers of hydroxyl radical or singlet oxygen, suggesting that the oxidant is superoxide anion. Inhibitors of various parts of the arachidonate cascade affect the TPA-induced oxidant response in a manner that corresponds to their effects on in vivo tumor promotion experiments. Agents that inhibit lipoxygenase activity, i.e. nordihydroguaiaretic acid, benoxaprofen, but not agents that are cyclooxygenase inhibitors, i.e. indomethacin, are effective in suppressing the oxidant response to TPA. Phospholipase C but not phospholipase A2 or D produced an oxidant response kinetically similar to that elicited by TPA. The inhibitors of TPA-induced oxidants inhibited the phospholipase C response to the same extent, suggesting that TPA and phospholipase C may produce an oxidant species through a common mechanism, via phospholipid turnover-protein kinase C activation. The relevance of oxidant production to the tumor promotion process is suggested by the ability of exogenous xanthine/xanthine oxidase, a superoxide anion-generating system, to induce ornithine decarboxylase, a characteristic of TPA-treated cells. In addition, oxidant production is significantly lower in cells from the TPA-promotion resistant C57BL/6J mouse. These studies provide further support for a role for reactive oxygens in the tumor promotion process.

Mitogen‐stimulated tyrosine phosphorylation of a 42‐kD cellular protein: Evidence for a protein kinase‐C requirement

Tyrosine phosphorylation of a 42-kD, cytosolic protein is a rapid consequence when quiescent cells are stimulated with any one of a diverse group of mitogenic agents. Among the inducers of this tyrosine phosphorylation are activators of protein kinase C, raising the possibility that this serine/threonine-specific protein kinase plays a role in mitogen-induced tyrosine phosphorylation. Using fibroblastic cells depleted of protein kinase C by chronic treatment with the tumor promoter tetradecanoyl phorbol acetate (TPA), we now show that protein kinase C is required for the tyrosine phosphorylation of the 42-kD protein, even when epidermal growth factor (EGF), whose receptor is a tyrosine-specific protein kinase, provides the initial stimulus. EGF is able to induce other cellular phosphorylations independent of protein kinase C, whereas thrombin appears to require the protein kinase C-dependent pathway. These findings suggest that phosphorylation of the 42-kD protein is part of a protein kinase C-dependent kinase cascade involved in intracellular signalling.

Oncogene transcription in normal human IMR-90 fibroblasts: Induction by serum and tetradecanoyl phorbol acetate

We report studies of oncogene transcription induced by the addition of serum to quiescent cultures of human IMR-90 fibroblasts. Oncogene messenger RNAs for c-myc, c-erbB and c-ras were increased in a specific temporal sequence after the addition of serum. Compounds that are proposed to exert their actions by the stimulation of cell growth were tested for their effect on oncogene transcription in IMR-90 fibroblasts. The tumor promoter tetradecanoyl phorbol acetate (TPA) was found to selectively induce the transcription of c-myc without observable effect on the transcription of the other oncogenes studied, and without inducing cell division. The inactive analog, phorbol didecanoate (PDD), and two complete carcinogens dimethylbenzathracene (DMBA) and 4-nitro quinoline-l-oxide (4NQO) were without effect on the transcription of the genes studied. These results suggest that the complete ordered sequence of gene transcription is necessary to achieve the physiologic response of cell division, and that classical promoters and complete carcinogens achieve their effects through different pathways.

Structural and functional studies of protein kinase C.

It has now become clear that protein kinase C (PKC) plays a fundamental role in cellular growth control in higher eukaryotes. In addition, since PKC has also been shown to be the high-affinity intracellular receptor for several classes of tumor promoters, the study of PKC has also become a central focus of current work in cancer research. We have isolated cDNA clones encoding several forms of this enzyme, and we have used these clones to begin to study in detail the role of PKC in growth control and tumor promotion. The complete primary structure of one of these forms, designated PKC beta 1, exhibits structural and functional characteristics which are shared among all of the currently identified forms of PKC. These include an amino terminal cysteine-rich domain which mediates Ca2+ and phospholipid binding, tumor promoter binding, and membrane association, and a carboxy terminal catalytic domain which possesses serine/threonine protein kinase activity. To further characterize the function of PKC, we have generated a series of rat fibroblast cell lines which stably overexpress a full-length cDNA encoding the beta 1 form of this enzyme. These cell lines contain a 20- to 53-fold increase in PKC activity, and also have an increase in high affinity phorbol ester receptors, relative to control cells. They also exhibit dramatically enhanced morphologic changes in response to treatment with the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA). These cell lines grow to a higher saturation density in monolayer culture and, when maintained at post-confluence, develop small, dense foci. In contrast to the control cells, which display complete anchorage dependence, the cell lines that overproduce PKC form small colonies in soft agar in the absence of TPA, and larger colonies in the presence of TPA. Thus, the mere overproduction of a single form of PKC is sufficient to confer anchorage independent growth and other growth abnormalities in rat fibroblasts. Taken together, these results provide direct evidence that PKC plays a critical role in normal cellular growth control and that it mediates several, and perhaps all of the cellular effects of the phorbol ester tumor promoters.

Studies on protein kinase C and colon carcinogenesis.

To further understand the molecular mechanisms of bile acid-mediated colon tumor promotion, we have examined the possible role of protein kinase C (PKC) in this process. Protein kinase C has been implicated in tumor promotion because it is the receptor for the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and mediates the action of this compound as well as that of other tumor promoters and growth factors. Our studies show that, in a manner analogous to 12-0-tetradecanoyl-phorbol-13-acetate, deoxycholic acid (DOA) can induce a time-dependent cellular redistribution of PKC as well as a concentration-dependent overexpression of the ornithine decarboxylase gene. These results taken together with our previous findings demonstrating decreased levels of PKC in human colon carcinomas compared with adjacent normal mucosa provide evidence that PKC has a role in colon carcinogenesis.

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.

Epidermal growth factor (GGF) and tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-0-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE 2 and 6-keto PGF 1α, an end metabolite of PGI 2. in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE 2 and 6-keto PGF 1α production; the maximum PG levels were obtained after 3–4 h incubation with EGF or TPA; the addition of as little as 10 −11 M EGF or 5×10 −11 M TPA resulted in increases in PGE 2 and 6-keto PGF 1α, and the maximum levels were obtained with 10 −8–10 −7 M EGF or TPA. This report also shows that EGF and TPA stimulate [ 3H] thymidine incorporation.

Phorbol ester, retinoic acid and diacylglycerol decrease ecto- but increase total basal-protein kinase activity of human fibroblasts

Studies have been carried out on intact human lung fibroblasts (HLF) in situ to investigate the effect of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) and the anti-tumor promoter retinoic acid (RA) on ecto-protein kinases. The ecto-kinase reaction of the HLF-cells was cAMP-independent, showed an apparent K m for ATP of 6.99 ± 0.35 (μM) and was substantially inhibited by TPA and RA. With the notable exception of a ∼ 57 kD phosphoprotein both compounds decreased the overall phosphorylation of intact cells. In contrast to RA-treatment, however, TPA caused the release of a high molecular weight (∼210 kD) phosphoprotein from the HLF. RA was the most potent retinoid in reducing ecto-kinase activity. The physiological modulators of protein kinase C: 1,2- and 1,3-diolein as well the synthetic 1,2-dioctanoylglycerol decreased the ecto-kinase activity to an extent similar to that of TPA. The drop in ecto-kinase activity of HLF-cells in situ caused by TPA, RA and the diacylglycerols was accompanied by an increase in total basal-(Mg ++-dependent) protein kinase activity present in extracts of treated cells. The results suggest an important role of ecto-kinase in the response of intact cells to TPA and RA.

Accumulation of activated mononuclear phagocytes in the liver following lipopolysaccharide treatment of rats.

Lipopolysaccharide (LPS) is a toxic bacterial cell wall component that is rapidly cleared from the portal circulation by Kupffer cells. To determine if interaction with LPS causes the accumulation and activation of mononuclear phagocytes (MNP) in the liver, we compared the morphological and functional characteristics of MNP obtained from livers of rats treated with LPS (5 mg/kg, intravenously [IV]) with normal resident Kupffer cells. MNP were isolated from rat livers by combined collagenase/pronase perfusion, selective digestion, and differential centrifugation on a metrizamide gradient. MNP obtained from livers of LPS-treated rats were found to display morphologic and functional characteristics of activated macrophages. These cells were generally larger than resident cells, were highly vacuolated, and adhered to culture dishes more rapidly. Both cell types phagocytized sheep red blood cells (sRBC) in a time-dependent manner, reaching a maximum after 60-75 min incubation with sRBC. However, MNP from livers of LPS-treated rats phagocytized 10-15 times more sRBC than resident Kupffer cells from untreated animals. Employing the Boyden chamber technique, both cell types were also found to be chemotactic to a number of stimuli including the complement fragment, C5a, the tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), and collagenous peptides related to tissue breakdown products. MNP from livers of LPS-treated rats were generally 10-15 times more responsive to the chemoattractants than resident Kupffer cells. In addition, both resident Kupffer cells and MNP from LPS-treated rats were found to release superoxide anion in response to stimulation by C5a and TPA. Taken together these results suggest that LPS treatment of rats leads to the recruitment and activation of MNP in the liver.

Differentiation of TERA-2 human embryonal carcinoma cells into neurons and HCMV permissive cells: Induction by agents other than retinoic acid

Retinoic acid induces the differentiation of NTERA-2 cl. D1 human embryonal carcinoma (EC) cells into neurons, cells permissive for the replication of human cytomegalovirus (HCMV), and other cell types that cannot as yet be classified but are distinguishable from the stem cells. We tested several additional agents for their ability to induce the differentiation of these EC cells. No differentiation was induced by butyrate, cyclic AMP, cytosine arabinoside, the tumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), or the chemotherapeutic agent cis-diaminedichloroplatinum, although morphological changes were detected at the highest concentrations of these agents that permitted cell survival. However, retinal, retinol, 5-bromouracil 2′deoxyribose (BUdR), 5-iodouracil 2′deoxyribose (IUdR), hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), and dimethylsulfoxide (DMSO) all induced some neuronal differentiation, but to a lesser extent than retinoic acid. Also, BUdR, IUdR, HMBA, and DMA induced the appearance of many cells permissive for the replication of HCMV. Differentiation was, in all cases, accompanied by the loss of SSEA-3, a globoseries glycolipid antigen characteristically expressed by human EC cells. However, another glycolipid antigen, A2B5, which appears in 60%–80% of differentiated cells 7 days following retinoic acid induction, was detected in less than 20% of the cells induced by the other agents studied. This implies that the HCMV-permissive cells induced by retinoic acid are not identical to those induced by BUdR, IUdR, and DMA.

Lack of inhibition of ultraviolet radiation-induced ornithine decarboxylase activity by retinoic acid.

Abstract— Normally present at low levels, ornithine decarboxylase (ODC) activity is induced to higher levels in animal skin by such disparate agents as tumor promoter 12–0‐tetradecanoylphorbol‐13‐acetate (TPA), UV radiation, and hair plucking. Retinoids are known to inhibit induction by TPA. Repeated applications of retinoic acid (RA) in acetone have also been reported to inhibit UV‐induced ODC in hairless mice. As a preliminary study, it was of interest to know whether RA in a cream vehicle would have the same effect. Groups of Skh‐hairless‐1 albino mice were irradiated once with Westinghouse FS‐20 lamps (0.045 Joules/cm/). Immediately post‐irradiation, RA was applied to the dorsum in different concentrations (0.001%, 0.002%, 0.02%), vehicles (cream and acetone) and on various time schedules (1–5 times). Sacrifice was by cervical dislocation 24 h later. Epidermis was obtained by mild heat separation and two epidermal sheets were pooled for each extract. In all experiments, the 70‐fold increase in UV‐induced ODC activity was further increased by retinoic acid by a factor ∼ 1.6. Since ODC levels are usually elevated in proliferating systems, the results are in concordance with the fact that both UV radiation and RA induce epidermal hyperplasia.

Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding.

Preincubation of Swiss 3T3 cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) at 37 degrees C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125I-platelet-derived growth factor (125I-PDGF), and does not affect the affinity of 125I-PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I-PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand-binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway.

Effects of the tumor promoter 12‐0‐tetradecanoyl‐phorbol‐13‐acetate on the wasp, Bracon hebetor

AbstractA single oral dose of the tumor promoter, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), caused a rapid necrosis of the ovarioles, aberrations in the developmental sequence of oocytes, and a concomitant dose‐dependent decline in egg production in the wasp, Bracon hebetor. TPA and its metabolities were found to have a biological half life of 26.7 h, with a peak concentration in the ovarioles in 3 h. Damage to ovariole tissue was persistent despite the relatively short half life. Other tissues in the wasp were largely unaffected, although TPA induced lethargy that persisted until death. There was no shortening of life span. Inhibition of intercellular transport and metabolic cooperation may account for decreased fecundity and fertility, but interaction with a phorbol ester receptor is more likely to account for developmental changes and central nervous system poisoning.

The effect of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) on the release of luteinizing hormone (LH) in dispersed rat anterior pituitary cells

The effect of the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) on the release of luteinizing hormone (LH) in dispersed rat anterior pituitary cells

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants.

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus-transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.

Endothelial Cells Degrade Extracellular Matrix Proteins Produced In Vitro

Bovine aortic endothelial cells (BAEC) were grown on extracellular matrices produced by vascular smooth muscle cells or fetal bovine endothelial cells. The glycoprotein components of these complex substrates were degraded through activation of the serum zymogen plasminogen to plasmin, as well as by a plasminogen independent protease(s). The plasminogen independent enzyme might be a protease with elastolytic activity since the BAEC digested elastin present in smooth muscle cell derived matrices. The cells also displayed collagenolytic activity on both types of matrices. The addition of the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) (10(-7) M) to the culture medium enhanced considerably the plasminogen activator and collagenolytic activities elaborated by BAEC resulting in an increased degradation rate of matrix glycoprotein and collagen components, whereas the elastolytic activity remained unaffected. Dexamethasone (10(-7) M), although suppressing plasminogen activator (PA) production by BAEC, did not alter their elastolytic activity allowing the cells to degrade the glycoprotein components of the matrices at an unchanged rate. The collagenolytic activity of the BAEC remained unaffected by dexamethasone. These studies demonstrate that BAEC elaborate different proteolytic enzyme activities allowing them to degrade various components of extracellular matrices. These enzymatic activities may be modulated by certain agents thus changing the degradative capabilities of the BAEC.

Quantitation of the intramembrane particle distribution in C3H/10T½ cells treated with TPA and retinyl acetate.

The mouse embryo fibroblast C3H/10T½ cells are used as a model system for the study of ultrastructural changes caused by tumor promoter and “anti-promoter” treatments. Noninitiated fibroblasts treated with tumor promoter undergo phenotypic transformation. On the other hand, the retinoid, retinyl acetate (RA), is effective in suppression of the malignant phenotype in the 10T½ cells (1). We report on the rapid freezing and freeze fracture electron microscopy study of the plasma membrane ultrastructures of noninitiated C3H/10T½ fibroblasts after treatments of retinyl acetate or a tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA).Rapid freezing of cells enables structure to be preserved without the use of cryoprotectants and chemical fixatives, which may alter membrane structural organization. For rapid freezing, 10T½ cells were grown as monolayers on glass specimen supports. The glass-copper specimen holder sandwiches were rapidly frozen by automatic plunging into liquid propane. Freeze fracture was carried out in a Polaron-Ultek ion pump apparatus with Cressington electron guns.