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Unlabelled: The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics., Methods: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts., Results: (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys., Conclusion: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A'' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A'' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A'' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A'' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.

Extensive protease digestion of delipidated [3H]mevalonate (MVA)-labeled proteins, followed by HPLC separation of the products, is one approach to identify and study prenyl cysteines. Using this methodology three major [3H]MVA-labeled peaks appeared. Two of them represent farnesyl cysteine (FC) and geranylgeranyl cysteine (GGC). The third peak represents unknown products that are considerably more hydrophobic than FC and GGC, here designated HPC. Previously, we provided evidence that cysteine residues may also be modified by dolichyl groups. Dolichyl cysteines (DolC) belong to HPC. However, as shown in the present study, DolC only represents a minor portion of HPC. Data obtained from different sets of experiments, including [3H]GGOH-labeling and use of prenyl transferase inhibitors, suggest that HPC mainly involves CXC or CC residues with double-linked GG groups. In turn this points to the possibility that proteins modified by double GG groups are quite common, and may probably involve other proteins than the rab family of GTPases., (Copyright 2001 Academic Press.)

Substantial evidence has suggested that a nonsterol product of mevalonic acid (MVA) is essential for the initiation of DNA synthesis in mammalian cells. Several possible isoprenoid candidates have been suggested, but the identity of this compound still remains unknown. In this study we have isolated and purified MVA products from SV40-transformed human fibroblasts and identified fractions with a growth-stimulatory effect. The cells were labelled with [14C]MVA in the presence of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. After lipid extraction, the [14C]MVA-labelled lipids were subjected to high performance liquid chromatography and size-exclusion chromatography, and the effect of the fractionated eluate on the DNA synthesis of arrested MVA-depleted target cells was tested. Thereby we found a fraction of [14C]MVA-labelled lipids with a substantial stimulatory effect on DNA synthesis. The chromatographic behavior suggested that the growth-stimulating fractions contained dolichol-20. This was confirmed by mass spectrometric analysis. Similar results were obtained when lipids from hepatocellular carcinoma cells and a sample from breast tumor were isolated and analyzed by the same procedure. The mechanisms by which these compounds induce DNA synthesis are unknown. Recent data obtained in our laboratory have provided evidence that dolichyl groups are covalently linked to tumor cell proteins, which implicates a new biological function for long-chain polyisoprenoid alcohols (Hjertman et al. [1997] FEBS Lett 416:235-238). In this study we demonstrate that tumor cells containing dolichol-like growth-stimulatory lipids also contained dolichylated proteins. This raises the question whether the growth-stimulatory dolichol-like lipids serve as substrates for the dolichylation reaction.

Labeling of human colon carcinoma cells with [3H]dol, followed by extensive delipidation and removal of dol-P oligosaccharides, showed that dol are bound to cellular proteins with sizes of 5, 10, 27, 75 and > 140 kDa. HPLC purification of proteolytic products of [3H]dol- and [35S]cys-labeled proteins revealed a hydrophobic peak containing both dol and cysteine. The dol/cys-labeled products were clearly separated from GG-cys, and exhibited a hydrophobicity between that of dol-P and dol. In another set of experiments delipidated proteins were treated with methyl iodide, which cleaves thioether bonds. After HPLC purification of released dol-like lipids, these were subjected to mass spectrometry. This demonstrated molecular ions with the same mass as that of dol. Taken together our data provide evidence for the existence of proteins covalently modified with dol.

One or more mevalonate derivatives of non-sterol type have been proposed to be of indispensable importance for cell growth. Conceivable mevalonate-dependent mechanisms involved in growth control are farnesylation of Ras proteins, regulation of c-myc expression, and N-linked glycosylation of the IGF-1 receptor. The latter mechanism might be rate-limited by dolichyl phosphate, which acts as a donor of oligosaccharides in glycoprotein synthesis in the endoplasmic reticulum. In order to study the significance for cell proliferation of the three aforementioned mevalonate-dependent processings, their inhibitory response due to mevalonate deprivation was explored and compared with the effect on DNA synthesis in the malignant melanoma cell line SK-MEL-2. We found that mevalonate depletion due to treatment with 3 microM lovastatin for 24 h, which efficiently growth-arrested the cells, hardly at all affected the expression of c-myc, and although Ras prenylation was inhibited by 50%, the most pronounced effect of lovastatin was seen on N-linked glycosylation of IGF-1 receptors, which was inhibited by more than 95%. The order and magnitude of the decreased IGF-1 receptor glycosylation, which was followed by a decreased expression of IGF-1 receptors at the cell membrane, correlated well with the inhibition of DNA synthesis. We investigated whether dolichol, and in particular dolichyl phosphate, through its participation in N-linked glycosylation, act as regulators of IGF-1 receptor expression. First, we could confirm that exogenous dolichol became phosphorylated and in this form took part in the glycosylation processing. Secondly, we showed that dolichyl phosphate, in a dose-dependent manner, could increase the number of IGF-1 receptors at the cell membrane, simultaneously as DNA synthesis was stimulated. Taken together, our results provide direct evidence for an important role of dolichyl phosphate as a regulator of cell growth through limiting N-linked glycosylation of the IGF-1 receptor.

Human diploid fibroblasts, arrested following serum or mevalonate depletion, were restimulated to a maximal rate of DNA synthesis within 24 h after the addition of serum or mevalonate, respectively. In both cases the initiation of DNA synthesis was preceded by a 12-h prereplicative phase. Upon the stimulation with serum there was a rapid increase in HMG-CoA reductase activity, reflecting an elevated formation of mevalonate, which reached its maximal value 4 h after serum replenishment. If this serum-induced increase in mevalonate synthesis was inhibited, the subsequent initiation of DNA synthesis was prevented. Serum stimulation also increased the level of N-linked glycosylation, an event that was dependent on the increase in HMG-CoA reductase activity. After treatment of the cells with tunicamycin, an inhibitor of N-linked glycosylation, they failed to enter the S-phase. However, an increased level of N-linked glycosylation was not required during the whole of the period after serum stimulation. Instead, it seemed to be of critical importance only during the mid stage of the prereplicative phase (i.e., 4-8 h after stimulation). Our data suggest that the N-linked glycosylation required for initiation of DNA synthesis is of high-molecular-weight (90-240 kDa) proteins. These high-molecular-weight glycoproteins may include growth factor receptors. Indirect evidence raises the possibility that the expression of growth factor receptors may play a regulatory role in the mevalonate-dependent growth activation of human fibroblasts.

Growth arrest induced by serum depletion and/or treatment with mevinolin (an inhibitor of mevalonate synthesis) in the human breast cancer cell line Hs578T was overcome by exogenous mevalonate, indicating that some product or metabolite of mevalonate may be involved in the mediation of serum-regulated growth of these cells. In the search for such compounds we first tested a variety of known end products of mevalonate with respect to their ability to counteract the inhibition of DNA synthesis caused by serum-free medium and mevinolin. Thereby high doses (10 micrograms/ml) of dolichol-20 were found to cause a partial counteraction. After straight-phase HPLC purification of endogenous lipids, isolated from 3H- or 14C-mevalonate-labelled Hs578T cultures, we found that non-sterol lipids co-eluting with dolichols efficiently induced DNA synthesis. After further purification with reverse-phase HPLC it was confirmed that virtually all of this effect was achieved by compounds(s) (seen as a single UV and radioactive peak) co-eluting with dolichol-20. Nanogram doses, at most, of this (these) compound(s) elicited a substantial stimulation of DNA synthesis. The lipid(s) also counteracted the inhibition by mevinolin of N-linked glycosylation, indicating that it (they) also interfere(s) with this processing. Since treatment with tunicamycin (an inhibitor of N-linked glycosylation) abolished this growth-stimulative effect, N-linked glycosylation seems to be a necessary event in the processes leading to lipid-induced initiation of DNA synthesis.