Name: measurement of heme synthesis levels in mammalian cells
Uploaded: 2015-07-09T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Heme Synthesis Levels in Mammalian Cells at JoVE.com

The HCC4017 and HBEC30KT cell lines were kindly provided by Dr. John Minna&#8217;s lab. This work was supported by the Cecil H. and Ida Green funds to Dr. Li Zhang.

CAUTION: While working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Dispose off all waste following local radiation safety guidelines.
1. Preparation of Cells
<ol>
	<li>Seed cells in 3.5 cm plates such that the confluency reaches 80%-90% on the day of assay.
	<ol>
		<li>Note that seeding confluency for cells depends upon the cell type and their growth rate. When treating cells with a reagent for a certain number of days, se...

Heme plays a key role in the generation of cellular energy via respiration26. Altered heme metabolism is known to be associated with various diseases including cancer28,41. Inhibition of heme synthesis is known to cause cell cycle arrest and apoptosis in Hela cells26,41. It has been shown that high heme synthesis level is associated with the progression of lung cancer cells28. Therefore, it would be of great importance to measure the levels of heme biosynthesis in cells under d...

Heme, a complex of ferrous iron and protoporphyrin IX is a central molecule for transporting and utilizing oxygen in virtually all living organisms1-3. The unique structure of heme enables it to function as a carrier of diatomic gases and electrons, as well as to perform various other functions1-5. For example, heme binds to oxygen in hemoglobin and myoglobin for the transfer and storage of oxygen6,7. It also works as an electron carrier in the cytochromes during respiration and acts as an electron donor for redox reactions catalyzed by cytochrome P450 enzymes8,9. One of the most significant features of heme is that, it can play regulatory roles in cellular and molecular processes such as gene transcription, protein synthesis and micro-RNA biogenesis4. For example, it affects the transcription of many genes by controlling the activity of mammalian transcriptional repressor Bach1 and the mammalian nuclear receptor Rev-erb&#945;10-15. Heme regulates the activation of heme activator protein (Hap) 1 which plays an important role in the activation of genes involved in respiration and controlling oxidative damage, in response to heme or oxygen16. Heme also regulates gene transcription in neuronal cells via nerve growth factor (NGF) signaling3,17-20. It also regulates protein synthesis in mammalian erythroid cells by modulating the activity of heme-regulated eIF2&#945; kinase (HRI)21-24. Furthermore, heme affects the activity of key signaling proteins such as tyrosine kinase Jak2 and Src, which are essential for proper cell functioning and cell growth4,20,25. It was found that in HeLa cells heme inhibition causes cell cycle arrest and activation of markers associated with senescence and apoptosis26. Both Heme deficiency or increased levels of heme are associated with severe health effects in humans27. Recent molecular and epidemiological studies have shown a positive association of high heme intake and increased risk of diseases, such as type-2 diabetes, coronary heart disease and several cancers including lung cancer, colorectal cancer and pancreatic cancer27,28. Using a matched pair of normal and cancer lung cells authors&#39; lab have found that cancer cells have increased levels of oxygen consumption, heme synthesis and proteins involved in heme uptake and oxygen utilization28. Interestingly, inhibition of heme synthesis decreased oxygen consumption, proliferation, migration and colony formation of cancer cells28. Thus, the fluctuation in the levels of endogenous heme plays an important role in the regulation of molecular and cellular processes3,4,28,29.
In mammals, the biosynthesis of heme occurs in eight steps, involving enzymes located in the mitochondria and the cytosol4 (Figure 1). Heme biosynthesis begins in the matrix of mitochondria with the condensation of glycine and succinyl-coA to form 5-aminolevulinic acid (5-ALA), catalyzed by ALA synthase (ALAS)4,31. This is the rate limiting step in heme biosynthesis in nonerythroid cells. 5-ALA is then exported out to the cytosol where the next four steps occur to form coproporphyrinogen III (CPgenIII), which is then imported back to the mitochondria, where it is converted into protoporphyrin IX (PPIX). Finally, one molecule of iron is incorporated to the protoporphyrin IX (PPIX) to produce heme, a reaction catalyzed by ferrochelatase (FECH)2,4.
The level of heme biosynthesis depends primarily on the level of ALAS enzyme which is tightly controlled by intracellular iron and heme4. The biosynthesis of heme can be affected by genetic defects, availability of certain minerals and vitamins (e.g., riboflavin, zinc), exposure to toxins (e.g., aluminum, lead), anoxia, fever, and levels of certain steroids (e.g., estrogen)32-35. The level of heme synthesis is altered in various diseased conditions. Decreased heme biosynthesis can cause anemia as well as neurological diseases3,36. Alternatively, increased heme biosynthesis plays an important role in the progression of certain cancers28,37. Heme has been shown to be critical for the growth, differentiation and survival of mammalian adipose, erythroid and neuronal cells4,38-41. For example, heme deficiency leads to neurite damage in primary mouse cortical neurons via the inhibition of glutamate NMDA (N-methyl-D-aspartate) receptor17. Additionally, inhibition of heme synthesis causes programmed cell death in the human epithelial cervix carcinoma HeLa cells26,41. Therefore, measuring the heme biosynthesis levels in various cells under different conditions is important for studying etiology and progression of many diseases.
Here we describe a fast and sensitive method to measure the level of intracellular heme synthesis by using [4-14C] 5-aminolevulic acid. This is an alternative method to other methods using 55Fe or 59Fe. We prefer using 14C because its radiation is very weak. In contrast, strong protection is required for working with Fe isotopes. Furthermore, this method is intended to measure and compare heme synthesis in different cells in parallel in a quick manner. In order to measure absolute heme levels, one may use the previously established method involving the use of HPLC42,43.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethy ether</td>
			<td>Sigma</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (Hydrochloric acid)</td>
			<td>Fisher</td>
			<td>A481-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Scintillation cocktail&#160;</td>
			<td>MP Biomedicals</td>
			<td>882470</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco</td>
			<td>15250</td>
			<td></td>
		</tr>
		<tr>
			<td>Radiolabeled 4-14C aminolevulinic acid</td>
			<td>Perkin-Elmer life sciences</td>
			<td></td>
			<td>Store @&#160; -80 &#176;C</td>
		</tr>
		<tr>
			<td>CelLytic M</td>
			<td>Sigma</td>
			<td>C2978</td>
			<td>Mammalian cell lysis reagent</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Specific reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Component</td>
			<td>Dispense</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Heme extraction buffer- Acetone: HCl:Water (25:1.3:5)</td>
			<td>Acetone</td>
			<td>25ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated HCl</td>
			<td>1.3ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>5ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of heme synthesis levels in mammalian cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

This method was used to compare the levels of heme synthesis in normal (HBEC30KT) vs. cancer (HCC4017) lung cells. Figure 2 shows a higher level of heme synthesis in cancer cells (HCC4017) than normal lung cells (HBEC30KT). The level of heme synthesis was also measured in normal and cancer cells in the presence of mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cells were treated with 10 &#956;M CCCP for 24 hr before the measurement of heme synthesis levels. As expected, the leve...

Watch this Scientific Journal Video about Measurement of Heme Synthesis Levels in Mammalian Cells at JoVE.com

Measurement of Heme Synthesis Levels in Mammalian Cells

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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