Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage.     

Proton and chloride currents in Chinese hamster ovary cells

Despite being one of the most frequent neoplasms occurring in the endocrine system, thyroid carcinoma is, nevertheless, a relatively rare event (0.5-1.5% of all malignant tumours in man); the differentiated forms are the most prevalent and are characterized by a high mean survival rate, whereas the very aggressive forms are rare and prognosis is unfavourable. Diagnostic evaluation of carcinomatous lesions, particularly in the early stages, may give rise to considerable difficulties at a clinical level due to the differentiation of the benign lesions, which are a frequent finding. The traditional clinico-semeiological and instrumental parameters, which, in the past, were used in the assessment of suspected malignancy, should not be considered as markers of malignancy; however, exposure to ionizing radiations during childhood may have a well defined role of risk. Following the recent progress in genetic and molecular studies, it is now possible to exploit genetic-molecular tumor markers and, at present, thyroid medullary carcinoma may be identified also in the absence of clinical evidence, particularly the familial form, thus allowing suitable prophylaxis in those subjects with specific genetic impairment (e.g. preventive thyroidectomy in infancy). Since no discriminating clinico-semeiological parameters are available, considering the aspecificity of scintigraphic findings and the lack of reliability of echographic imaging in providing data which enable us to distinguish a rare neoplastic pattern from the more frequent finding of a benign thyroid mass, fine-needle aspiration (FNA) cytology may today be considered the technique of choice in the screening of the thyroid nodule. Our experience in over 12,000 nodular lesions since 1982, has confirmed that the cytological examination is the most discriminating investigation, diagnostic reliability being far greater than that of traditional techniques. Considering the high frequency of thyroid nodule disease which rarely harbours a carcinomatous lesion, a very scrupulous diagnostic algorithm is mandatory. The FNA cytology, together with morphofunctional and immunological examinations, as well as dynamic exploration of the thyroid hypothalamo-pituitary axis, which allows a nosographic picture of the thyroid nodule disease, provides a more discriminating appraisal for the surgical approach to a single, solitary or prominent nodule.     

Antiapoptotic signaling by the insulin receptor in Chinese hamster ovary cells

We have sought to determine whether insulin can promote cell survival and protect Chinese hamster ovary (CHO) cells from apoptosis induced by serum starvation. Low concentrations of insulin were antiapoptotic for cells overexpressing wild-type insulin receptors but not in cells transfected with kinase-defective insulin receptor mutants that lacked a functional ATP binding site. However, treatment with orthovanadate (50 microM), a widely used tyrosine phosphatase inhibitor, led a dramatic reduction in internucleosomal DNA fragmentation in both cell lines. Cells transfected with truncated receptor mutants in either the juxtamembrane or C-terminal domain were as responsive as cells overexpressing wild-type receptors in mediating insulin antiapoptotic protection. The mechanisms underlying insulin antiapoptotic protection were investigated using a variety of pharmacological tools known to inhibit distinct signaling pathways. The phosphatidylinositol-3' kinase inhibitors wortmannin and LY294002 had only a modest influence whereas blocking protein farnesylation with manumycin severely disrupted the antiapoptotic capacity of the insulin receptor. Of interest, cells gained antiapoptotic potential following inhibition of extracellular signal-regulated kinase activation with the pharmacological agent PD98059. Insulin induced MKK3/MKK6 phosphorylation and activation of p38 MAP kinase whose activity was inhibited with SB203580. However, the inhibition of p38 MAP kinase had no effect on the protection offered by insulin. We conclude that the antiapoptotic function of the insulin receptor requires intact receptor kinase activity and implicates a farnesylation-dependent pathway. Increase in cellular phosphotyrosine content, however, triggers antiapoptotic signal that may converge downstream of the insulin receptor.     

Apoptosis in batch cultures of Chinese hamster ovary cells

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis--as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.     

Shrinkage-induced protein tyrosine phosphorylation in Chinese hamster ovary cells

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (approximately 42, approximately 85, and approximately 120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosM), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl- levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.     

Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT- gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates "dual inheritance" at the APRT locus in CHO cells.     

Modulation of membrane drug permeability in Chinese hamster ovary cells

The kinetics of colchicine uptake into Chinese hamster ovary cells have been investigated and found to be consistent with an unmediated diffusion mode. A variety of compounds such as local anesthetics and non-ionic detergents as well as drugs such as vinblastine, vincristine, daunomycin and actinomycin D potentiate the rate of colchicine uptake into these cells and into colchicine resistant mutants. In all cases, higher concentrations of these compounds were required to stimulate colchicine uptake in the colchicine resistant mutants than in the cells of the parental line. This stimulation was observed also in the uptake of puromycin, a structurally and functionally different drug. These stimulatory agents did not, however, cause the cells to become nonspecifically leaky since the uptake of 2-deoxy-D-glucose was unaffected. In addition, the activation energy of colchicine uptake was unaltered in the presence of stimulating agents, implying that they were not causing colchicine to enter the cells via a different mechanism. The results are compatible with the view that these compounds are membrane-active, and are able to stimulate an increased rate of unmediated diffusion of colchicine into the cells. It appears that a mechanism for the regulation of passive permeability is modified in the resistant mutants.     

Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro

G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures.     

Cytosar-U (Ara-C) induced changes in mouse jejunal crypt epithelial kinetics and radiosensitivity to gamma rays and fast neutrons

Pretreatment with the S phase specific cytotoxic agent Cytosine Arabinoside (Ara-C) protects the intestinal stem cells from gamma radiation injury by nearly tenfold. Studies were undertaken to test whether an altered cell age distribution could account for the reported duration and degree of Ara-C induced protection and to measure the degree of protection from the high energy neutrons of the Fermilab Cancer Treatment Facility. Twelve hours after treatment with Ara-C, B6CF1/ANL mice were exposed to increasing single doses of either 137Cs gamma-rays or neutrons from the Fermilab accelerator, or a split dose of neutrons with intervals of 1, 2, and 3 hours. The number of regenerating microcolonies per jejunal circumference in Ara-C treated and irradiated animals was compared to irradiated controls. Another group of mice was given Ara-C but in the 12-hour interval between Ara-C and irradiation, colcemid was given every 3 hours to continuously block and kill cells in mitosis. The results suggest that Ara-C given 12 hours prior to neutron irradiation protects intestinal stem cells to nearly the same degree as it does from 137Cs gamma-ray damage. Furthermore, the control split-dose recovery ratio to neutron irradiation at 1, 2, or 3 hours was 1.8 and was unchanged 12 hours after Ara-C. Colcemid reduced the crypt cell population to less than half the normal 250 cells per crypt; however, the cell survival curve was unaltered from the survival curve 12 hours after Ara-C. These results suggest that Ara-C recruits intestinal clonogenic stem cells, but inhibits their normal passage through DNA synthesis. These cells, responsible for intestinal mucosal regeneration, appear to be held in a radioresistant portion of the cell cycle for a period of about 10-16 hours after Ara-C.     

Ca2+ triggers massive exocytosis in Chinese hamster ovary cells

We have tracked the cell surface area of CHO cells by measuring the membrane capacitance, Cm. An increase in cytosolic [Ca2+], [Ca2+]i, increased the cell surface area by 20-30%. At micromolar [Ca2+]i the increase occurred in minutes, while at 20 microM or higher [Ca2+]i it occurred in seconds and was transient. GTPgammaS caused a 3% increase even at 0.1 microM [Ca2+]i. We conclude that CHO cells, previously thought capable only of constitutive exocytosis, can perform Ca2+-triggered exocytosis that is both massive and rapid. Ca2+-triggered exocytosis was also observed in 3T3 fibroblasts. Our findings add evidence to the view that Ca induces exocytosis in cells other than known secretory cells.     

Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells

Methylglyoxal is an alpha-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 microM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 microM, 0.02% of cellular amino acid residues could be modified. As few as one or two "hits" with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.     

Activation of phospholipase A2 by the nociceptin receptor expressed in Chinese hamster ovary cells

To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca2+-dependent cytosolic PLA2 (cPLA2) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA2, suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX-sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

Characterization of brain-type ryanodine receptor permanently expressed in Chinese hamster ovary cells

To clarify a function of brain-type ryanodine receptor (RyR3) and its regulation, we established a stable cell line expressing rabbit RyR3 by transfection of Chinese hamster ovary cells (CHO cells) with the cDNA and investigated characteristics of the RyR3. Scatchard analysis of [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 showed two distinct binding sites. The Kd values of high and low affinity binding sites were 1.92 and 25.9 nM, respectively. [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 was dependent on pCa. Extracellular Ca2+ (2-10 mM) and high concentration (more than 30 mM) of caffeine activated the RyR3 in CHO cells and increased its intracellular Ca2+ concentration. The enhancement of [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 was observed by bromoeudistomin D (BED), a caffeine-like powerful Ca2+ releaser, at pCa 5.5. Stably expressed RyR3 in CHO is useful for characterization of its function.     

Frequencies and types of exchange aberrations induced by X-rays and neutrons in Chinese hamster splenocytes detected by FISH using chromosome-specific DNA libraries

PURPOSE: To estimate the frequencies of radiation- (low and high LET) induced chromosome aberrations in Chinese hamster splenocytes by two-colour fluorescence in situ hybridization using DNA painting probes specific for chromosomes 2, 3, 8, X and Y and to determine (1) the ratio of radiation-induced translocations and dicentrics; (2) the spectrum of exchange aberrations induced by X-rays and neutrons; and (3) the relative involvement of the different chromosomes in the formation of aberrations. MATERIALS AND METHODS: Isolated splenocytes from the Chinese hamster were irradiated in vitro with different doses of 200 kV X-rays (0.75, 1.5, 3.0 Gy) and 1 MeV fast neutrons (0.25, 0.5, 1.0 Gy). Conventional analysis of chromosome aberrations was carried out in Giemsa-stained preparations. Chromosome aberrations involving chromosomes 2, 3, 8, X and Y were analysed in first division metaphases using two-colour FISH. RESULTS: The results indicate that when all types of translocations are taken into account both X-rays and neutrons induce more translocations than dicentrics, the ratio between the two types of exchanges being 1.4 and 1.8 respectively. The ratio of 'apparently simple' reciprocal translocations and reciprocal complete dicentrics was close to 1 for both types of radiation. The RBE of neutrons for induction of exchanges was found to be between 5 and 8. Neutron irradiation was more efficient at inducing insertions. Among the chromosomes studied, an increased involvement was observed for chromosome 8 in dicentrics and translocations than that expected on the basis of its chromosome length. The high content of interstitial telomeric sequences in chromosome 8 may be responsible for the observed sensitivity of this chromosome. CONCLUSIONS: The results obtained in this study indicate that: (1) more translocations are found than dicentrics; (2) heterogeneity exists among Chinese hamster chromosomes for involvement in radiation-induced exchanges; (3) the spectrum and distribution of exchange aberrations are different between X-rays and neutrons; and (4) the relative frequencies of insertions could be used as a 'fingerprint' for exposure to high LET radiation.     

Activation of aniline by extracts from plants and induction of chromosomal damages in Chinese hamster ovary cells

Activation of aniline by plant extracts was studied by a chromosomal damage induction assay in Chinese hamster ovary (CHO) cells in vitro. Extracts from roots of Vicia faba activated aniline and the activation caused increases in chromosomal aberrations (CAs) and endoreduplicated cells (ERCs), but did not cause sister-chromatid exchanges (SCEs). Extracts from Pisum sativum and Lactuca sativa, however, did not activate aniline. All C-hydroxylated metabolites of aniline, o-aminophenol, m-aminophenol and p-aminophenol, induced not only CAs but also SCEs in CHO cells. These results show that the pathway for aniline activation by Vicia extracts is by means other than the C-hydroxylation.     

Mcl-1, a member of the Bcl-2 family, delays apoptosis induced by c-Myc overexpression in Chinese hamster ovary cells

Mcl-1, a protein increased early in the differentiation of human myeloblastic ML-1 cells, has sequence similarity to Bcl-2. In the present study, we determined whether Mcl-1 has functional similarity to Bcl-2 by testing its ability to inhibit apoptosis induced by c-Myc overexpression. This was carried out using Chinese hamster ovary 5AHSmyc cells which contain the human c-myc proto-oncogene under the control of a heat shock promoter. Heat treatment induces c-Myc overexpression and thus apoptosis as determined by internucleosomal DNA fragmentation. We transfected 5AHSmyc cells with mcl-1 and found that clones expressing the introduced Mcl-1 protein exhibited reduced DNA fragmentation. Mcl-1 was also capable of delaying the onset of cell death as judged by loss of membrane integrity, although it could not provide complete protection from c-Myc overexpression. Thus, Mcl-1 has functional homology to Bcl-2 in that Mcl-1 can enhance cell viability under conditions that otherwise cause apoptosis.     

The effect of oxidative stress on the production of the recombinant protein, interferon gamma, produced by Chinese hamster ovary cells in stirred-batch culture

BACKGROUND: The uptake of thallium-201 (T1-201) in malignant tumors is associated with malignant potential (metastatic potential and proliferative activity). The grade of accumulation of T1-201 in malignant tumors may provide information regarding prognosis. METHODS: A T1-201 single photon emission computed tomography was conducted 120 minutes after intravenous injection of 111 megabecquerel of T1-201 chloride. The authors calculated the uptake ratio to evaluate the degree of T1-201 uptake in the primary tumor. This ratio was compared with survival time and other prognostic factors. RESULTS: The authors studied 152 patients (125 men and 27 women). The group of patients with the low T1-201 uptake ratio survived longer than the group of patients with the high T1-201 uptake ratio (median survival, 58 weeks vs. 33 weeks; P = 0.0138 by the log rank test). The multivariate analysis confirmed that the T1-201 uptake ratio was an independent prognostic factor for survival. CONCLUSIONS: These results suggest that the T1-201 uptake ratio provides independent and objective prognostic information for patients with lung carcinoma.     

Spontaneous and induced mutagenesis in a culture of Chinese hamster cells

Spontaneous and nitrosoguanidine (NG)-induced rate of reversions to glutamine independence was studied in cultured temperature-sensitive glutamine auxotrophs of Chinese hamster cells. In 3 experiments the spontaneous rate of reversions varied from 0.8-10(-6) to 3.84-10(-6) per cell per generation. A dependence of the yield of NG-induced back mutations upon the time interval between the mutagenic treatment and the transfer to selective conditions (glutamine deficient medium, 40 degrees C) was established. No induced revertants were detected when cells were transferred to selective conditions immediately after the treatment with NG. After 2--3 days cultivation in glutamine containing medium at 36 degrees C and the sunsequent transfer to selective conditions the frequency of induced reversions varied from 0.56-10(-4) to 10.55-10(-4) in different experiments; after 6 days -- from 0.05-10(-4) to 4.0-10(-4). In all cases where induction was detected, the difference, between the frequency of glutamine prototrophs in treated and control plates was significant. Glutamine independence proved to be stable after prolonged cultivation under non-selective conditions, the degree of prototrophy being greatly unequal in different clones. No differnce in this respect was detected between spontaneous and NG-induced revertants. The proposed system of reverse mutations can be used for studying diverse problems of somatic cell genetics.     

Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells

To determine the presence and identity of isopentenyladenosine-containing transfer RNAs (tRNAs) in a mammalian cell line, we adopted a novel method to isolate, clone and sequence these RNAs. This method was based on 3' polyadenylation of the tRNA prior to cDNA synthesis, PCR amplification, cloning and DNA sequencing. Using this unique procedure, we report the cloning and sequencing of the selenocysteine-tRNA and mitochondrial tryptophan-tRNA from Chinese hamster ovary cells which contain this specific tRNA modification. This new method will be useful in the identification of other tRNAs and other small RNAs where the primary sequence is unknown.     

Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells

The biosynthesis of the prohormone convertase PC2 was studied in Chinese hamster ovary cells stably transfected with PC2 cDNA (CHO/PC2) and in rat insulinoma cells (Rin5f). The major form of PC2 synthesized by CHO/PC2 cells was a 75-kDa protein corresponding to proPC2; this protein was retained intracellularly for 2-4 h following synthesis, suggesting prolonged intracellular residence. In contrast, the major form of PC2 within Rin cells initially exhibited a molecular mass of 72 kDa and was then progressively converted to a 64-kDa species. This 64-kDa species, which required 1-2 h to be released, was the major PC2 form detectable in Rin cell medium. Calcium-dependent benzyloxycarbonyl-Arg-Ser-Lys-Arg-aminomethylcoumarin cleaving activity was found in spent Rin cell medium; this activity could be immunoprecipitated with a carboxyl-terminal PC2 antibody, but not with preimmune serum. In neither cell line did intracellular PC2 become endoglycosidase H-resistant over time. PC2 released from Rin cells was also endoglycosidase H-sensitive. Microsequencing and endoglycosidase H results indicate that 75-kDa CHO cell PC2 and 72-kDa Rin cell PC2 both represent proPC2. We speculate that (a) PC2 undergoes unusual glycosylation, which may be related to its slow release from cells, and (b) the 64-kDa molecule detectable in spent Rin cell medium represents the enzymatically active form of PC2.